Genes involved in isoprenoid compound production

ABSTRACT

Genes have been isolated from Methylomonas 16a sp. encoding the isoprenoid biosynthetic pathway. The genes and gene products are the first isolated from a Methylomonas strain that is capable of utilizing single carbon (C1) substrates as energy sources. The genes and gene products of the present invention may be used in a variety of ways for the production of isoprenoid compounds in a variety of organisms.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/229,907, filed Sep. 1, 2001.

FIELD OF THE INVENTION

[0002] This invention is in the field of microbiology. Morespecifically, this invention pertains to nucleic acid fragments encodingenzymes useful for microbial production of isoprenoid compounds.

BACKGROUND OF THE INVENTION

[0003] Isoprenoids are an extremely large and diverse group of naturalproducts that have a common biosynthetic origin, i.e., a singlemetabolic precursor, isopentenyl diphosphate (IPP). The group of naturalproducts known as isoprenoids includes all substances that are derivedbiosynthetically from the 5-carbon compound isopentenyl diphosphate.Isoprenoid compounds are also referred to as “terpenes” or “terpenoids”,which is the term used in the designation of the various classes ofthese examples (Spurgeon and Porter, Biosynthesis of IsoprenoidCompounds, pp 346, A Wiley-Interscience Publication (1981)).

[0004] Isoprenoids are ubiquitous compounds found in all livingorganisms. Some of the well-known examples of isoprenoids are steroids(triterpenes), carotenoids (tetraterpenes), and squalene, just to name afew.

[0005] For many years, it was accepted that IPP was synthesized throughthe well-known acetate/mevalonate pathway. However, recent studies havedemonstrated that the mevalonate-dependent pathway does not operate inall living organisms. An alternate mevalonate-independent pathway forIPP biosynthesis was initially characterized in bacteria and later alsoin green algae and higher plants (Horbach et al., FEMS Microbiol. Lett.111:135-140 (1993); Rohmer et al, Biochem. 295: 517-524 (1993);Schwender et al., Biochem. 316: 73-80 (1996); Eisenreich et al., Proc.Natl. Acad. Sci. USA 93: 6431-6436 (1996)).

[0006] Many steps in both the mevalonate-independent andmevalonate-dependent isoprenoid pathways are known. For example, theinitial steps of the alternate pathway involve the condensation of3-carbon molecules (pyruvate and C1 aldehyde group, D-glyceraldehyde3-phosphate), to yield the 5-carbon compoundD-1-deoxyxylulose-5-phosphate. Lois et al. has reported a gene, dxs,that encodes D-1-deoxyxylulose-5-phosphate synthase (DXS) and thatcatalyzes the synthesis of D-1-deoxyxylulose-5-phosphate in E. coli(Proc. Natl. Acad. Sci. USA 95: 2105-2110 (1998)).

[0007] Next, the isomerization and reduction ofD-1-deoxyxylulose-5-phosphate yields2-C-methyl-D-erythritol-4-phosphate. One of the enzymes involved in theisomerization and reduction process is D-1-deoxyxylulose-5-phosphatereductoisomerase (DXR). Takahashi et al. reported that the dxr geneproduct catalyzes the formation of 2-C-methyl-D-erythritol-4-phosphatein the alternate pathway in E. coli (Proc. Natl. Acad. Sci. USA 95:9879-9884 (1998)).

[0008] Steps converting 2-C-methyl-D-erythritol-4-phosphate toisopentenyl monophosphate are not well characterized, although somesteps are known. 2-C-methyl-D-erythritol-4-phosphate is converted into4-diphosphocytidyl-2C-methyl-D-erythritol in a CTP dependent reaction bythe enzyme encoded by non-annotated gene ygbP. Rohdich et al. reportedthat the YgbP protein in E. coli catalyzes the reaction mentioned above.Recently, ygbP gene was renamed as ispD as a part of the isp genecluster (SwissProt #Q46893) (Proc. Natl. Acad. Sci. USA 96:11758-11763(1999)).

[0009] Then the 2 position hydroxy group of4-diphosphocytidyl-2C-methyl-D-erythritol can be phosphorylated in anATP dependent reaction by the enzyme encoded by the ychB gene. Luttgenet al. has reported that the YchB protein in E. coli phosphorylates4-diphosphocytidyl-2C-methyl-D-erythritol, resulting in4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate. Recently, theychB gene was renamed as ispE as a part of the isp gene cluster(SwissProt#P24209) (Luttgen et al., Proc. Natl. Acad. Sci. USA97:1062-1067 (2000)).

[0010] Herz et al. reported that the ygbB gene product in E. coliconverts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate to2C-methyl-D-erythritol 2,4-cyclodiphosphate. 2C-methyl-D-erythritol2,4-cyclodiphosphate can be further converted into carotenoids throughthe carotenoid biosynthesis pathway (Proc. Natl. Acad. Sci. USA97:2486-2490 (2000)). Recently, the ygbB gene was renamed as ispF as apart of the isp gene cluster (SwissProt #P36663).

[0011] The reaction catalyzed by the YgbP enzyme is carried out in a CTPdependent manner. Thus, CTP synthase plays an important role in theisoprenoid pathway. PyrG encoded by the pyrG gene in E. coli wasdetermined to encode CTP synthase (Weng et al., J. Biol. Chem.,261:5568-5574 (1986)).

[0012] Following several reactions not yet characterized, isopentenylmonophosphate is formed. Isopentenyl monophosphate is converted toisopentenyl diphosphate (IPP) by isopentenyl monophosphate kinase,encoded by the ipk gene, and that is identical to the above mentionedyhcB (ispE) gene (Lange and Croteau, Proc. Natl. Acad. Sci. USA96:13714-13719 (1999)).

[0013] Cunningham et al. (J of Bacteriol. 182:5841-5848, (2000)) hasreported that the lytB gene in E. coli that is thought to encode anenzyme of the deoxyxylulose-5-phosphate pathway that catalyzes a step ator subsequent to the point at which the pathway branches to form IPP anddimethylallyl diphosphate. LytB gene is also found in othermicroorganisms such as Acinetbacter and Synechocystis, (GenBankAccession Numbers AF027189 and U38915, respectively).

[0014] Prenyltransferases constitute a broad group of enzymes catalyzingthe consecutive condensation of isopentenyl diphosphate (IPP) resultingin the formation of prenyl diphosphates of various chain lengths.Homologous genes of prenyl transferase have highly conserved regions intheir amino acid sequences. Ohto et al. reported three prenyltransferase genes in cyanobacterium Synechococcus elongatus (Plant Mol.Biol. 40:307-321 (1999)). They are geranylgeranyl (C20) diphosphatesynthase, farnesyl (C15) diphosphate synthase and anotherprenyltransferase that can catalyze the synthesis of five prenyldiphosphates of various lengths.

[0015] Further down in the isoprenoid biosynthesis pathway, more genesare involved in the synthesis of specific isoprenoids. As an example,the crtN gene was found in Heliobacillus mobilis (Xiang et al., Proc.Natl. Acad. Sci. USA 95:14851-14856 (1998)) to encode diapophytoenedehydrogenase is a part of the carotenoid biosynthesis pathway.

[0016] Although most of the genes involved in the isoprenoid pathwaysare known, the genes involved in the isoprenoid pathway ofmethanotrophic bacteria are not described in the existing literature.However, there are many pigmented methylotrophic and methanotrophicbacteria, which suggests that the ability to produce carotenoid pigmentsis widespread in these bacteria and therefore the genes must bewidespread in these bacteria. Applicants have isolated a number ofunique open reading frames encoding enzymes of the isoprenoidbiosynthesis pathway from a Methylomonas sp.

[0017] Applicants have solved the stated problem by isolating genescontaining 9 open reading frames (ORFs) encoding enzymes involved inisoprenoid synthesis.

SUMMARY OF THE INVENTION

[0018] The present invention provides an isolated nucleic acid moleculeencoding a isoprenoid biosynthetic enzyme, selected from the groupconsisting of: (a) an isolated nucleic acid molecule encoding the aminoacid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6,8, 10, 12, 14, 16, 18 and 24; (b) an isolated nucleic acid molecule thathybridizes with (a) under the following hybridization conditions:0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS; and (c) an isolated nucleic acid molecule that iscomplementary to (a) or (b).

[0019] Additionally the invention provides polypeptides encoded by thepresent genes and chimera where the genes are under the control ofsuitable regulatory sequences. Similarly the invention providestransformed organisms, including bacteria, yeast, filamentous fungi, andgreen plants expressing one or more of the present genes and geneproducts.

[0020] The present invention provides methods of obtaining all orsubstantial portions of the instant genes through gene amplification orhybridization methods.

[0021] In another embodiment the invention provides methods for theproduction of isoprenoids comprising: contacting a transformed host cellunder suitable growth conditions with an effective amount of a carbonsource whereby an isoprenoid compound is produced, said transformed hostcell comprising a set of nucleic acid molecules encoding SEQ ID NOs:2,4, 6, 8, 10, 12, 14, 16, 18, and 24 under the control of suitableregulatory sequences.

[0022] Similarly the invention provides a method of regulatingisoprenoid biosynthesis in an organism comprising, over-expressing atleast one isoprenoid gene selected from the group consisting of SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17 and 23 in an organism such that theisoprenoid biosynthesis is altered in the organism.

[0023] In another embodiment the invention provides a mutated geneencoding a isoprenoid enzyme having an altered biological activityproduced by a method comprising the steps of (i) digesting a mixture ofnucleotide sequences with restriction endonucleases wherein said mixturecomprises:

[0024] a) a native isoprenoid gene;

[0025] b) a first population of nucleotide fragments which willhybridize to said native isoprenoid gene;

[0026] c) a second population of nucleotide fragments which will nothybridize to said native isoprenoid gene;

[0027] wherein a mixture of restriction fragments are produced; (ii)denaturing said mixture of restriction fragments; (iii) incubating thedenatured said mixture of restriction fragments of step (ii) with apolymerase; (iv) repeating steps (ii) and (iii) wherein a mutatedisoprenoid gene is produced encoding a protein having an alteredbiological activity.

BRIEF DESCRIPTION OF THE DRAWINGS, SEQUENCE DESCRIPTIONS, AND THEBIOLOGICAL DEPOSITS

[0028]FIG. 1 shows the isoprenoid pathway.

[0029]FIG. 2 shows two gene clusters contain genes in the isoprenoidpathway. One cluster contains the ispD, ispF and pyrG genes, and theother cluster contains the crtN1 and crtN2 genes.

[0030]FIG. 3a shows a gene dose effect on carotenoid biosynthesis. Twocultures of the native strain of Methylomonas 16a, designated as 16a,and two cultures of a rif-resistant variant of the native strain,designated as 16a-rif (without plasmid), served as negative controls.Six isolated transconjugants were labeled as DXP-1 through DPX-6. FIG.3b shows the plasmid that contains the dxs and dxr genes.

[0031] The invention can be more fully understood from the followingdetailed description and the accompanying sequence descriptions, whichform a part of this application.

[0032] The following sequences comply with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

[0033] SEQ ID NO:1 is the nucleotide sequence of ORF 1 encoding the dxsgene.

[0034] SEQ ID NO:2 is the deduced amino acid sequence of dxs encodedbyORF1.

[0035] SEQ ID NO:3 is the nucleotide sequence of ORF 2 encoding the dxrgene.

[0036] SEQ ID NO:4 is the deduced amino acid sequence of dxr encoded byORF 2.

[0037] SEQ ID NO:5 is the nucleotide sequence of ORF 3 encoding the ygbP(ispD) gene.

[0038] SEQ ID NO:6 is the deduced amino acid sequence of ygbP (ispD)gene encoded by ORF 3.

[0039] SEQ ID NO:7 is the nucleotide sequence of ORF 4 encoding the ychB(ispE) gene.

[0040] SEQ ID NO:8 is the deduced amino acid sequence of ychB (ispE)encoded by ORF 4.

[0041] SEQ ID NO:9 is the nucleotide sequence of ORF 5 encoding the ygbB(ispF) gene.

[0042] SEQ ID NO:10 is the deduced amino acid sequence of ygbB (ispF)encoded by ORF 5.

[0043] SEQ ID NO:11 is the nucleotide sequence of ORF 6 encoding thepyrG gene.

[0044] SEQ ID NO:12 is the deduced amino acid sequence of pyrG encodedby ORF 6.

[0045] SEQ ID NO:13 is the nucleotide sequence of ORF 7 encoding theispA gene.

[0046] SEQ ID NO:14 is the deduced amino acid sequence of ispA geneencoded by ORF 7.

[0047] SEQ ID NO:15 is the nucleotide sequence of ORF 8 encoding thecrtN gene, copyl.

[0048] SEQ ID NO:16 is the deduced amino acid sequence of crtN genecopyl encoded by ORF 8.

[0049] SEQ ID NO:17 is the nucleotide sequence of ORF 9 encoding thecrtN gene copy2.

[0050] SEQ ID NO:18 is the deduced amino acid sequence of crtN genecopy2 encoded by ORF 9.

[0051] SEQ ID NO:19 and 20 are the primer sequences used to amplify thedxs gene.

[0052] SEQ ID NO:21 and 22 are the primer sequences used to amplify thedxr gene.

[0053] SEQ ID NO:23 is the nucleotide sequence of ORF 10 encoding theIytB gene.

[0054] SEQ ID NO:24 is the deduced amino acid sequence of the IytB geneencoded by ORF 10.

[0055] Applicants made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for the Purposes of Patent Procedure: InternationalDepositor Identification Depository Reference Designation Date ofDeposit Methylomonas 16a ATCC PTA 2402 August 21 2000

DETAILED DESCRIPTION OF THE INVENTION

[0056] The genes and their expression products are useful for thecreation of recombinant organisms that have the ability to producevarious isoprenoid compounds. Nucleic acid fragments encoding the abovementioned enzymes have been isolated from a strain of Methylomonas 16aand identified by comparison to public databases containing nucleotideand protein sequences using the BLAST and FASTA algorithms well known tothose skilled in the art.

[0057] The genes and gene products of the present invention may be usedin a variety of ways for the enhancement or manipulation of isoprenoidcompounds.

[0058] The microbial isoprenoid pathway is naturally a multi-productplatform for production of compounds such as carotenoids, quinones,squalene, and vitamins. These natural products may be from 5 carbonunits to more than 55 carbon units in chain length. There is a generalpractical utility for microbial isoprenoid production for carotenoidcompounds as these compounds are very difficult to make chemically(Nelis and Leenheer, Appl. Bacteriol. 70:181-191 (1991)). Mostcarotenoids have strong color and can be viewed as natural pigments orcolorants. Furthermore, many carotenoids have potent antioxidantproperties and thus inclusion of these compounds in the diet is thoughtto healthful. Well-known examples are β-carotene and astaxanthin.

[0059] In the case of Methylomonas 16a, the inherent capacity to producecarotenoids is particularly useful. This is because methanotrophicbacteria have been used for the commercial production of single cellprotein and the protein from these bacteria is known to be efficaciousas animal feeds (Green, Taxonomy of Methylotrophic Bacteria. In: Methaneand Methanol Utilizers (Biotechnology Handbooks 5) J. Colin Murrell andHoward Dalton eds. 1992 Pleanum Press NY. Pp 23-84; BioProteinManufacture 1989. Ellis Horwood series in applied science and industrialtechnology. NY: Halstead Press.)

[0060] The genes and gene sequences described herein enable one toincorporate the production of healthful carotenoids directly into thesingle cell protein product derived from Methylomonas 16a. This aspectmakes this strain or any methanotrophic strain into which these genesare incorporated a more desirable production host for animal feed due tothe presence of carotenoids which are known to add desirablepigmentation and health benefits to the feed. Salmon and shrimpaquacultures are particularly useful applications for this invention ascarotenoid pigmentation is critically important for the value of theseorganisms. (F. Shahidi, J. A. Brown, Carotenoid pigments in seafood andaquaculture Critical reviews in food Science 38(1): 1-67 (1998)).

[0061] In addition to feed additives, the genes are useful for theproduction of carotenoids and their derivatives, isoprenoidintermediates and their derivatives, and as pure products useful aspigments, flavors and fragrances.

[0062] In this disclosure, a number of terms and abbreviations are used.The following definitions are provided.

[0063] “Open reading frame” is abbreviated ORF.

[0064] “Polymerase chain reaction” is abbreviated PCR.

[0065] As used herein, an “isolated nucleic acid fragment” is a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

[0066] The term “isoprenoid” or “terpenoid” refers to any moleculederived from the isoprenoid pathway including 10-carbon terpenoids andtheir derivatives, such as carotenoids and xanthophylls.

[0067] The term “Methylomonas 16a” and “Methylomonas 16a sp.” are usedinterchangeably and refer to the Methylomonas strain used in the presentinvention.

[0068] The term “Dxs” refers to the 1-deoxyxylulose-5-phosphate synthaseenzyme encoded by the dxs gene represented in OFR1.

[0069] The term “Dxr” refers to the 1-deoxyxylulose-5-phosphatereductoisomerase enzyme encoded by the dxr gene represented in ORF 2.

[0070] The term “YgbP” or “IspD” refers to the 2C-methyl-D-erythritolcytidyltransferase enzyme encoded by the ygbP or ispD gene representedin ORF 3. The names of the gene, ygbP or ispD, are used interchangeablyin this application. The names of gene product, YgbP or IspD are usedinterchangeably in this application.

[0071] The term “YchB” or “IspE” refers to4-diphosphocytidyl-2-C-methylerythritol kinase enzyme encoded by ychB orispE gene represented in ORF 4. The names of the gene, ychB or ispE, areused interchangeably in this application. The names of gene product,YchB or IspE are used interchangeably in this application.

[0072] The term “YgbB” or “IspF” refers to the 2C-methyl-d-erythritol2,4-cyclodiphosphate synthase enzyme encoded by the ygbB or ispF generepresented in ORF 5. The names of the gene, ygbB or ispF, are usedinterchangeably in this application. The names of gene product, YgbB orIspF are used interchangeably in this application.

[0073] The term “PyrG” refers to the CTP synthase enzyme encoded by thepyrG gene represented in ORF 6.

[0074] The term “IspA” refers to the geranyltransferase or farnesyldiphosphate synthase enzyme, as one of the prenyl transferase familyencoded by the ispA gene represented in ORF 7.

[0075] The term “CrtN1” or “CrtN, copyl” refers to copy 1 of thediapophytoene dehydrogenase enzymeencoded by the crtN1 gene representedin ORF 8.

[0076] The term “CrtN2” or “CrtN copy2” refers to copy 2 of thediapophytoene dehydrogenase enzymeencoded by the crtN2 gene representedin ORF 9.

[0077] The term “LytB” refers to the protein encoded by the IytB generepresented in ORF 10, functioning in the formation of IPP anddimethylallyl diphosphate in the isoprenoid pathway.

[0078] The term “Embden-Meyerhof pathway” refers to the series ofbiochemical reactions for conversion of hexoses such as glucose andfructose to important cellular 3-carbon intermediates such asglyceraldehyde 3 phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate and pyruvate. These reactions typically proceed with net yieldof biochemically useful energy in the form of ATP. The key enzymesunique to the Embden-Meyerof pathway are the phosphofructokinase andfructose 1,6 bisphosphate aldolase.

[0079] The term “Entner-Douderoff pathway” refers to a series ofbiochemical reactions for conversion of hexoses such as as glucose orfructose to the important 3 carbon cellular intermediates pyruvate andglyceraldehyde 3 phosphate without any net production of biochemicallyuseful energy. The key enzymes unique to the Entner-Douderoff pathwayare the 6 phosphogluconate dehydratase and a ketodeoxyphosphogluconatealdolase.

[0080] The term “high growth methanotrophic bacterial strain” refers toa bacterium capable of growth with methane or methanol as the solecarbon and energy source and which possess a functional Embden-Meyerofcarbon flux pathway resulting in a high rate of growth and yield of cellmass per gram of C1 substrate metabolized. The specific “high growthmethanotrophic bacterial strain” described herein is referred to as“Methylomonas 16a” or “16a”, which terms are used interchangeably.

[0081] The term “methanotroph” or “methanotrophic bacteria” will referto a prokaryotic microorganism capable of utilizing methane as itsprimary carbon and energy source.

[0082] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the protein encoded by the DNA sequence.“Substantially similar” also refers to nucleic acid fragments whereinchanges in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression byantisense or co-suppression technology. “Substantially similar” alsorefers to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotide basesthat do not substantially affect the functional properties of theresulting transcript. It is therefore understood that the inventionencompasses more than the specific exemplary sequences.

[0083] For example, it is well known in the art that alterations in agene which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded protein are common. For the purposes of the present inventionsubstitutions are defined as exchanges within one of the following fivegroups:

[0084] 1. Small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr (Pro, Gly);

[0085] 2. Polar, negatively charged residues and their amides: Asp, Asn,Glu, Gln;

[0086] 3. Polar, positively charged residues: His, Arg, Lys;

[0087] 4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys);and

[0088] 5. Large aromatic residues: Phe, Tyr, Trp.

[0089] Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue (such as glycine) or a more hydrophobic residue (such as valine,leucine, or isoleucine). Similarly, changes which result in substitutionof one negatively charged residue for another (such as aspartic acid forglutamic acid) or one positively charged residue for another (such aslysine for arginine) can also be expected to produce a functionallyequivalent product.

[0090] In many cases, nucleotide changes which result in alteration ofthe N-terminal and C-terminal portions of the protein molecule wouldalso not be expected to alter the activity of the protein.

[0091] Each of the proposed modifications is well within the routineskill in the art, as is determination of retention of biologicalactivity of the encoded products. Moreover, the skilled artisanrecognizes that substantially similar sequences encompassed by thisinvention are also defined by their ability to hybridize, understringent conditions (0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC,0.1% SDS followed by 0.1×SSC, 0.1% SDS), with the sequences exemplifiedherein. Preferred substantially similar nucleic acid fragments of theinstant invention are those nucleic acid fragments whose DNA sequencesare at least 80% identical to the DNA sequence of the nucleic acidfragments reported herein. More preferred nucleic acid fragments are atleast 90% identical to the DNA sequence of the nucleic acid fragmentsreported herein. Most preferred are nucleic acid fragments that are atleast 95% identical to the DNA sequence of the nucleic acid fragmentsreported herein.

[0092] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 andTable 11.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C. Hybridization requires that the two nucleic acids containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of Tm for hybrids of nucleicacids having those sequences. The relative stability (corresponding tohigher Tm) of nucleic acid hybridizations decreases in the followingorder: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100nucleotides in length, equations for calculating Tm have been derived(see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorternucleic acids, i.e., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra, 11.7-11.8). In oneembodiment the length for a hybridizable nucleic acid is at least about10 nucleotides. Preferable a minimum length for a hybridizable nucleicacid is at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least 30 nucleotides.Furthermore, the skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as length of the probe.

[0093] A “substantial portion” of an amino acid or nucleotide sequencecomprising enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nim.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches partial or completeamino acid and nucleotide sequences encoding one or more particularmicrobial proteins. The skilled artisan, having the benefit of thesequences as reported herein, may now use all or a substantial portionof the disclosed sequences for purposes known to those skilled in thisart. Accordingly, the instant invention comprises the complete sequencesas reported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0094] The term “complementary” is used to describe the relationshipbetween nucleotide bases that are capable to hybridizing to one another.For example, with respect to DNA, adenosine is complementary to thymineand cytosine is complementary to guanine. Accordingly, the instantinvention also includes isolated nucleic acid fragments that arecomplementary to the complete sequences as reported in the accompanyingSequence Listing as well as those substantially similar nucleic acidsequences.

[0095] The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing:Informatics and Genome Proiects (Smith, D. W., ed.) Academic Press, NY(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, NY (1991). Preferred methods to determine identity are designedto give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0096] Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein. Suitable nucleic acidfragments not only have the above homologies but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids.

[0097] “Codon degeneracy” refers to the nature in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment that encodes all or asubstantial portion of the amino acid sequence encoding the instantmicrobial polypeptides as set forth in SEQ ID NOs. The skilled artisanis well aware of the “codon-bias” exhibited by a specific host cell inusage of nucleotide codons to specify a given amino acid. Therefore,when synthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

[0098] “Synthetic genes” can be assembled from oligonucleotide buildingblocks that are chemically synthesized using procedures known to thoseskilled in the art. These building blocks are ligated and annealed toform gene segments which are then enzymatically assembled to constructthe entire gene. “Chemically synthesized”, as related to a sequence ofDNA, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of DNA may be accomplished usingwell-established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the genes can be tailored for optimal gene expression basedon optimization of nucleotide sequence to reflect the codon bias of thehost cell. The skilled artisan appreciates the likelihood of successfulgene expression if codon usage is biased towards those codons favored bythe host. Determination of preferred codons can be based on a survey ofgenes derived from the host cell where sequence information isavailable.

[0099] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0100] “Coding sequence” refers to a DNA sequence that codes for aspecific amino acid sequence. “Suitable regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing site, effector binding site andstem-loop structure.

[0101] “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

[0102] The “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

[0103] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO9928508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated yet hasan effect on cellular processes.

[0104] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone is affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

[0105] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide.

[0106] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0107] The term “signal peptide” refers to an amino terminal polypeptidepreceding the secreted mature protein. The signal peptide is cleavedfrom and is therefore not present in the mature protein. Signal peptideshave the function of directing and translocating secreted proteinsacross cell membranes. Signal peptide is also referred to as signalprotein.

[0108] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

[0109] The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

[0110] The term “altered biological activity” will refer to an activity,associated with a protein encoded by a microbial nucleotide sequencewhich can be measured by an assay method, where that activity is eithergreater than or less than the activity associated with the nativemicrobial sequence. “Enhanced biological activity” refers to an alteredactivity that is greater than that associated with the native sequence.“Diminished biological activity” is an altered activity that is lessthan that associated with the native sequence.

[0111] The term “sequence analysis software” refers to any computeralgorithm or software program that is useful for the analysis ofnucleotide or amino acid sequences. “Sequence analysis software” may becommercially available or independently developed. Typical sequenceanalysis software will include but is not limited to the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison,Wis. 53715 USA), and the FASTA program incorporating the Smith-Watermanalgorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int.Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor.Publisher: Plenum, New York, N.Y.). Within the context of thisapplication it will be understood that where sequence analysis softwareis used for analysis, that the results of the analysis will be based onthe “default values” of the program referenced, unless otherwisespecified. As used herein “default values” will mean any set of valuesor parameters which originally load with the software when firstinitialized.

[0112] Standard recombinant DNA and molecular cloning techniques usedhere are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M.L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

[0113] Sequence Identification

[0114] A variety of nucleotide sequences have been isolated fromMethylomonas 16a encoding gene products involved in the isoprenoidproduction pathway. ORF's 1-6 for example encode enzymes early in theisoprenoid pathway (FIG. 1) leading to IPP, which is the precursor ofall isoprenoid compounds. ORF 7 encodes the IspA enzyme that is involvedin elongation by condensing IPP precursors. ORF 8 and ORF 9 are involvedmore specifically in carotenoid production.

[0115] Comparison of the dxs nucleotide base and deduced amino acidsequences (ORF 1) to public databases reveals that the most similarknown sequences range from about 60% identical to the amino acidsequence of reported herein over length of 620 amino acids using aSmith-Waterman alignment algorithm (W. R. Pearson, Comput. MethodsGenome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992,111-20.Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.). Morepreferred amino acid fragments are at least about 80%-90% identical tothe sequences herein. Most preferred are nucleic acid fragments that areat least 95% identical to the amino acid fragments reported herein.Similarly, preferred Dxs encoding nucleic acid sequences correspondingto the instant ORF's are those encoding active proteins and which are atleast 80% identical to the nucleic acid sequences of reported herein.More preferred Dxs nucleic acid fragments are at least 90% identical tothe sequences herein. Most preferred are Dxs nucleic acid fragments thatare at least 95% identical to the nucleic acid fragments reportedherein.

[0116] Comparison of the Dxr base and deduced amino acid sequence topublic databases reveals that the most similar known sequence is 55%identical at the amino acid level over a length of 394 amino acids (ORF2) using a Smith-Waterman alignment algorithm (W. R. Pearson supra).More preferred amino acid fragments are at least about 80%-90% identicalto the sequences herein. Most preferred are nucleic acid fragments thatare at least 95% identical to the amino acid fragments reported herein.Similarly, preferred Dxr encoding nucleic acid sequences correspondingto the instant ORF are those encoding active proteins and which are atleast 80% identical to the nucleic acid sequences of reported herein.More preferred Dxr nucleic acid fragments are at least 90% identical tothe sequences herein. Most preferred are Dxr nucleic acid fragments thatare at least 95% identical to the nucleic acid fragments reportedherein.

[0117] Comparison of the YgbP (IspD) base and deduced amino acidsequences to public databases reveals that the most similar knownsequences range from about 52% identical at the amino acid level over alength of 231 amino acids (ORF 3) using a Smith-Waterman alignmentalgorithm (W. R. Pearson supra). More preferred amino acid fragments areat least about 80%-90% identical to the sequences herein. Most preferredare nucleic acid fragments that are at least 95% identical to the aminoacid fragments reported herein. Similarly, preferred YgbP (IspD)encoding nucleic acid sequences corresponding to the instant ORF arethose encoding active proteins and which are at least 80% identical tothe nucleic acid sequences of reported herein. More preferred YgbP(IspD) nucleic acid fragments are at least 90% identical to thesequences herein. Most preferred are YgbP (IspD) nucleic acid fragmentsthat are at least 95% identical to the nucleic acid fragments reportedherein.

[0118] Comparison of the YchB (IspE) base and deduced amino acidsequences to public databases reveals that the most similar knownsequences range from about 50% identical at the amino acid level over alength of 285 amino acids (ORF 4) using a Smith-Waterman alignmentalgorithm (W. R. Pearson supra). More preferred amino acid fragments areat least about 80%-90% identical to the sequences herein. Most preferredare nucleic acid fragments that are at least 95% identical to the aminoacid fragments reported herein. Similarly, preferred YchB (IspE)encoding nucleic acid sequences corresponding to the instant ORF arethose encoding active proteins and which are at least 80% identical tothe nucleic acid sequences of reported herein. More preferred YchB(IspE) nucleic acid fragments are at least 90% identical to thesequences herein. Most preferred are YchB (IspE) nucleic acid fragmentsthat are at least 95% identical to the nucleic acid fragments reportedherein.

[0119] Comparison of the YgbB (IspF) base and deduced amino acidsequences to public databases reveals that the most similar knownsequences range from about 69% identical at the amino acid level over alength of 157 amino acids (ORF 5) using a Smith-Waterman alignmentalgorithm (W. R. Pearson supra). More preferred amino acid fragments areat least about 80%-90% identical to the sequences herein. Most preferredare nucleic acid fragments that are at least 95% identical to the aminoacid fragments reported herein. Similarly, preferred YgbB (IspF)encoding nucleic acid sequences corresponding to the instant ORF arethose encoding active proteins and which are at least 80% identical tothe nucleic acid sequences of reported herein. More preferred YgbB(IspF) nucleic acid fragments are at least 90% identical to thesequences herein. Most preferred are YgbB (IspF) nucleic acid fragmentsthat are at least 95% identical to the nucleic acid fragments reportedherein.

[0120] Comparison of the PyrG base and deduced amino acid sequences topublic databases reveals that the most similar known sequences rangefrom about 67% identical at the amino acid level over a length of 544amino acids (ORF 6) using a Smith-Waterman alignment algorithm (W. R.Pearson supra). More preferred amino acid fragments are at least about80%-90% identical to the sequences herein. Most preferred are nucleicacid fragments that are at least 95% identical to the amino acidfragments reported herein. Similarly, preferred PyrG encoding nucleicacid sequences corresponding to the instant ORF are those encodingactive proteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred PyrG nucleic acid fragmentsare at least 90% identical to the sequences herein. Most preferred arePyrG nucleic acid fragments that are at least 95% identical to thenucleic acid fragments reported herein.

[0121] Comparison of the IspA base and deduced amino acid sequences topublic databases reveals that the most similar known sequences rangefrom about 57% identical at the amino acid level over a length of 297amino acids (ORF 7) using a Smith-Waterman alignment algorithm (W. R.Pearson supra). More preferred amino acid fragments are at least about80%-90% identical to the sequences herein. Most preferred are nucleicacid fragments that are at least 95% identical to the amino acidfragments reported herein. Similarly, preferred IspA encoding nucleicacid sequences corresponding to the instant ORF are those encodingactive proteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred IspA nucleic acid fragmentsare at least 90% identical to the sequences herein. Most preferred areIspA nucleic acid fragments that are at least 95% identical to thenucleic acid fragments reported herein.

[0122] Comparison of the copy 1 of CrtN base and deduced amino acidsequences to public databases reveals that the most similar knownsequences range from about 34% identical at the amino acid level over alength of 511 amino acids (ORF 8) using a Smith-Waterman alignmentalgorithm (W. R. Pearson supra). More preferred amino acid fragments areat least about 80%-90% identical to the sequences herein. Most preferredare nucleic acid fragments that are at least 95% identical to the aminoacid fragments reported herein. Similarly, preferred nucleic acidsequences corresponding to the instant ORF are those encoding activeproteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred nucleic acid fragments areat least 90% identical to the sequences herein. Most preferred arenucleic acid fragments that are at least 95% identical to the nucleicacid fragments reported herein.

[0123] Comparison of the copy 2 of CrtN base and deduced amino acidsequences to public databases reveals that the most similar knownsequences range from about 34% identical at the amino acid level over alength of 497 amino acids (ORF 9) using a Smith-Waterman alignmentalgorithm (W. R. Pearson supra). More preferred amino acid fragments areat least about 80%-90% identical to the sequences herein. Most preferredare nucleic acid fragments that are at least 95% identical to the aminoacid fragments reported herein. Similarly, preferred nucleic acidsequences corresponding to the instant ORF are those encoding activeproteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred nucleic acid fragments areat least 90% identical to the sequences herein. Most preferred arenucleic acid fragments that are at least 95% identical to the nucleicacid fragments reported herein.

[0124] Comparison of the LytB base and deduced amino acid sequences topublic databases reveals that the most similar known sequences rangefrom a about 65% identical at the amino acid level over a length of 318amino acids (ORF 10) using a Smith-Waterman alignment algorithm (W. R.Pearson supra). It has been reported that expression of IytB gene in E.coli significantly enhanced accumulation of carotenoids when the E. coliwas engineered to express carotenoid (Cunningham et al., J of Bacteriol.182:5841-5848 (2000)). More preferred amino acid fragments are at leastabout 80%-90% identical to the sequences herein. Most preferred arenucleic acid fragments that are at least 95% identical to the amino acidfragments reported herein. Similarly, preferred nucleic acid sequencescorresponding to the instant ORF are those encoding active proteins andwhich are at least 80% identical to the nucleic acid sequences ofreported herein. More preferred nucleic acid fragments are at least 90%identical to the sequences herein. Most preferred are nucleic acidfragments that are at least 95% identical to the nucleic acid fragmentsreported herein.

[0125] Isolation of Homologs

[0126] The nucleic acid fragments of the instant invention may be usedto isolate genes encoding homologous proteins from the same or othermicrobial species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g. polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No.4,683,202), ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad.Sci. USA 82, 1074, (1985)) or strand displacement amplification (SDA,Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89, 392, (1992)).

[0127] For example, genes encoding similar proteins or polypetides tothose of the instant invention could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired bacteria using methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primers DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part of or full-length of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length DNA fragments under conditionsof appropriate stringency.

[0128] Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art. (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986)pp. 33-50 IRL Press, Herndon, Va.); Rychlik, W. (1993) In White, B. A.(ed.), Methods in Molecular Biology, Vol. 15, pages 31-39, PCRProtocols: Current Methods and Applications. Humania Press, Inc.,Totowa, N.J.)

[0129] Generally two short segments of the instant sequences may be usedin polymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding microbial genes.

[0130] Alternatively, the second primer sequence may be based uponsequences derived from the cloning vector. For example, the skilledartisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998(1988)) to generate cDNAs by using PCR to amplify copies of the regionbetween a single point in the transcript and the 3′ or 5′ end. Primersoriented in the 3′ and 5′ directions can be designed from the instantsequences. Using commercially available 3′ RACE or 5′ RACE systems(BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al.,PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).

[0131] Alternatively the instant sequences may be employed ashybridization reagents for the identification of homologs. The basiccomponents of a nucleic acid hybridization test include a probe, asample suspected of containing the gene or gene fragment of interest,and a specific hybridization method. Probes of the present invention aretypically single stranded nucleic acid sequences which are complementaryto the nucleic acid sequences to be detected. Probes are “hybridizable”to the nucleic acid sequence to be detected. The probe length can varyfrom 5 bases to tens of thousands of bases, and will depend upon thespecific test to be done. Typically a probe length of about 15 bases toabout 30 bases is suitable. Only part of the probe molecule need becomplementary to the nucleic acid sequence to be detected. In addition,the complementarity between the probe and the target sequence need notbe perfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

[0132] Hybridization methods are well defined. Typically the probe andsample must be mixed under conditions which will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration theshorter the hybridization incubation time needed. Optionally achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature [Van Ness and Chen (1991) Nucl. Acids Res.19:5143-5151]. Suitable chaotropic agents include guanidinium chloride,guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate,sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, andcesium trifluoroacetate, among others. Typically, the chaotropic agentwill be present at a final concentration of about 3M. If desired, onecan add formamide to the hybridization mixture, typically 30-50% (v/v).

[0133] Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1 Mbuffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pH rangeabout 6-9), about 0.05 to 0.2% detergent, such as sodium dodecylsulfate,or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500kilodaltons), polyvinylpyrrolidone (about 250-500 kdal), and serumalbumin. Also included in the typical hybridization solution will beunlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmentednucleic DNA, e.g., calf thymus or salmon sperm DNA, or yeast RNA, andoptionally from about 0.5 to 2% wt./vol. glycine. Other additives mayalso be included, such as volume exclusion agents which include avariety of polar water-soluble or swellable agents, such as polyethyleneglycol, anionic polymers such as polyacrylate or polymethylacrylate, andanionic saccharidic polymers, such as dextran sulfate.

[0134] Nucleic acid hybridization is adaptable to a variety of assayformats. One of the most suitable is the sandwich assay format. Thesandwich assay is particularly adaptable to hybridization undernon-denaturing conditions. A primary component of a sandwich-type assayis a solid support. The solid support has adsorbed to it or covalentlycoupled to it immobilized nucleic acid probe that is unlabeled andcomplementary to one portion of the sequence.

[0135] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening DNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen DNA expression libraries toisolate full-length DNA clones of interest (Lemer, R. A. Adv. Immunol.36:1 (1984); Maniatis).

[0136] Recombinant Expresion—Microbial

[0137] The genes and gene products of the instant sequences may beproduced in heterologous host cells, particularly in the cells ofmicrobial hosts. Expression in recombinant microbial hosts may be usefulfor the expression of various pathway intermediates, or for themodulation of pathways already existing in the host for the synthesis ofnew products heretofore not possible using the host.

[0138] Preferred heterologous host cells for expression of the instantgenes and nucleic acid fragments are microbial hosts that can be foundbroadly within the fungal or bacterial families and which grow over awide range of temperature, pH values, and solvent tolerances. Forexample, it is contemplated that any bacteria, yeast, and filamentousfungi will be suitable hosts for expression of the present nucleic acidfragments. Because of transcription, translation and the proteinbiosynthetic apparatus is the same irrespective of the cellularfeedstock, functional genes are expressed irrespective of carbonfeedstock used to generate cellular biomass. Large-scale microbialgrowth and functional gene expression may utilize a wide range of simpleor complex carbohydrates, organic acids and alcohols, and/or saturatedhydrocarbons such as methane or carbon dioxide in the case ofphotosynthetic or chemoautotrophic hosts. However, the functional genesmay be regulated, repressed or depressed by specific growth conditions,which may include the form and amount of nitrogen, phosphorous, sulfur,oxygen, carbon or any trace micronutrient including small inorganicions. In addition, the regulation of functional genes may be achieved bythe presence or absence of specific regulatory molecules that are addedto the culture and are not typically considered nutrient or energysources. Growth rate may also be an important regulatory factor in geneexpression. Examples of host strains include but are not limited tofungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces,Pichia, Candida, Hansenula, or bacterial species such as Salmonella,Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia,Pseudomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus,Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Anabaena,Thiobacillus, Methanobacterium and Klebsiella.

[0139] Of particular interest in the present invention are high growthobligate methanotrophs having an energetically favorable carbon fluxpathway. For example Applicants have discovered a specific strain ofmethanotroph having several pathway features which make it particularlyuseful for carbon flux manipulation. This type of strain has served asthe host in the present application and is known as Methylomonas 16a (ATCC PTA 2402).

[0140] The present strain contains several anomalies in the carbonutilization pathway. For example, based on genome sequence data, thestrain is shown to contain genes for two pathways of hexose metabolism.The Entner-Douderoff Pathway which utilizes the keto-deoxyphosphogluconate aldolase enzyme is present in the strain. It isgenerally well accepted that this is the operative pathway in obligatemethanotrophs. Also present, however, is the Embden-Meyerhof Pathway,which utilizes the fructose bisphosphate aldolase enzyme. It is wellknown that this pathway is either not present or not operative inobligate methanotrophs. Energetically, the latter pathway is mostfavorable and allows greater yield of biologically useful energy,ultimately resulting in greater yield production of cell mass and othercell mass-dependent products in Methylomonas 16a. The activity of thispathway in the present 16a strain has been confirmed through microarraydata and biochemical evidence measuring the reduction of ATP. Althoughthe 16a strain has been shown to possess both the Embden-Meyerhof andthe Entner-Douderoff pathway enzymes, the data suggests that theEmbden-Meyerhof pathway enzymes are more strongly expressed than theEntner-Douderoff pathway enzymes. This result is surprising and counterto existing beliefs concerning the glycolytic metabolism ofmethanotrophic bacteria. Applicants have discovered other methanotrophicbacteria having this characteristic, including for example, Methylomonasclara and Methylosinus sporium. It is likely that this activity hasremained undiscovered in methanotrophs due to the lack of activity ofthe enzyme with ATP, the typical phosphoryl donor for the enzyme in mostbacterial systems.

[0141] A particularly novel and useful feature of the Embden-Meyerhofpathway in strain 16a is that the key phosphofructokinase step ispyrophosphate dependent instead of ATP dependent. This feature adds tothe energy yield of the pathway by using pyrophosphate instead of ATP.Because of its significance in providing an energetic advantage to thestrain, this gene in the carbon flux pathway is considered diagnosticfor the present strain.

[0142] In methanotrophic bacteria methane is converted to biomoleculesvia a cyclic set of reactions known as the ribulose monophosphatepathway or RuMP cycle. This pathway is comprised of three phases, eachphase being a series of enzymatic steps. The first step is “fixation” orincorporation of C-1 (formaldehyde) into a pentose to form a hexose orsix-carbon sugar. This occurs via a condensation reaction between a5-carbon sugar (pentose) and formaldehyde and is catalyzed by hexulosemonophosphate synthase. The second phase is termed “cleavage” andresults in splitting of that hexose into two 3-carbon molecules. One ofthose 3-carbon molecules is recycled back through the RuMP pathway andthe other 3-carbon fragment is utilized for cell growth. Inmethanotrophs and methylotrophs the RuMP pathway may occur as one ofthree variants. However only two of these variants are commonly found:the FBP/TA (fructose bisphosphotase/Transaldolase) or the KDPG/TA (ketodeoxy phosphogluconate/transaldolase) pathway (Dijkhuizen L., G. E.Devries. The Physiology and biochemistry of aerobic methanol-utilizinggram negative and gram positive bacteria. In: Methane and MethanolUtilizers 1992, ed Colin Murrell and Howard Dalton Plenum Press NY).

[0143] The present strain is unique in the way it handles the “cleavage”steps where genes were found that carry out this conversion via fructosebisphosphate as a key intermediate. The genes for fructose bisphosphatealdolase and transaldolase were found clustered together on one piece ofDNA. Secondly the genes for the other variant involving the keto deoxyphosphogluconate intermediate were also found clustered together.Available literature teaches that these organisms (obligatemethylotrophs and methanotrophs) rely solely on the KDPG pathway andthat the FBP-dependent fixation pathway is utilized by facultativemethylotrophs (Dijkhuizen et al., supra). Therefore the latterobservation is expected whereas the former is not. The finding of theFBP genes in an obligate methane utilizing bacterium is both surprisingand suggestive of utility. The FBP pathway is energetically favorable tothe host microorganism due to the fact that more energy (ATP) isutilized than is utilized in the KDPG pathway. Thus organisms thatutilize the FBP pathway may have an energetic advantage and growthadvantage over those that utilize the KDPG pathway. This advantage mayalso be useful for energy-requiring production pathways in the strain.By using this pathway a methane-utilizing bacterium may have anadvantage over other methane utilizing organisms as production platformsfor either single cell protein or for any other product derived from theflow of carbon through the RuMP pathway.

[0144] Accordingly the present invention provides a method for theproduction of an isopreoid compound in a high growth, energeticallyfavorable Methylomonas strain which

[0145] (a) grows on a Cl carbon substrate selected from the groupconsisting of methane and methanol; and

[0146] (b) comprises a functional Embden-Meyerhof carbon pathway, saidpathway comprising a gene encoding a pyrophosphate dependentphosphofructokinase enzyme.

[0147] Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of the any of thegene products of the instant sequences. These chimeric genes could thenbe introduced into appropriate microorganisms via transformation toprovide high level expression of the enzymes

[0148] Accordingly it is expected, for example, that introduction ofchimeric genes encoding the instant bacterial enzymes under the controlof the appropriate promoters, will demonstrate increased isoprenoidproduction. It is contemplated that it will be useful to express theinstant genes both in natural host cells as well as heterologous host.Introduction of the present genes into native host will result inelevated levels of existing isoprenoid production. Additionally, theinstant genes may also be introduced into non-native host bacteria wherethere are advantages to manipulate the isoprenoid compound productionthat are not present in Methanotrophs.

[0149] Vectors or cassettes useful for the transformation of suitablehost cells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

[0150] Initiation control regions or promoters, which are useful todrive expression of the instant ORF's in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1,PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful forexpression in Saccharomyces); AOX1 (useful for expression in Pichia);and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc (useful forexpression in Escherichia coli) as well as the amy, apr, npr promotersand various phage promoters useful for expression in Bacillus.

[0151] Termination control regions may also be derived from variousgenes native to the preferred hosts. Optionally, a termination site maybe unnecessary, however, it is most preferred if included.

[0152] Pathway Engineering

[0153] Knowledge of the sequence of the present genes will be useful inmanipulating the isoprenoid biosynthetic pathways in any organism havingsuch a pathway and particularly in methanotrophs. Methods ofmanipulating genetic pathways are common and well known in the art.Selected genes in a particularly pathway may be upregulated or downregulated by variety of methods. Additionally, competing pathwaysorganism may be eliminated or sublimated by gene disruption and similartechniques.

[0154] Once a key genetic pathway has been identified and sequencedspecific genes may be upregulated to increase the output of the pathway.For example, additional copies of the targeted genes may be introducedinto the host cell on multicopy plasmids such as pBR322. Alternativelythe target genes may be modified so as to be under the control ofnon-native promoters. Where it is desired that a pathway operate at aparticular point in a cell cycle or during a fermentation run, regulatedor inducible promoters may used to replace the native promoter of thetarget gene. Similarly, in some cases the native or endogenous promotermay be modified to increase gene expression. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,PCT/US93/03868).

[0155] Alternatively it may be necessary to reduce or eliminate theexpression of certain genes in the target pathway or in competingpathways that may serve as competing sinks for energy or carbon. Methodsof down-regulating genes for this purpose have been explored. Wheresequence of the gene to be disrupted is known, one of the most effectivemethods gene down regulation is targeted gene disruption where foreignDNA is inserted into a structural gene so as to disrupt transcription.This can be effected by the creation of genetic cassettes comprising theDNA to be inserted (often a genetic marker) flanked by sequence having ahigh degree of homology to a portion of the gene to be disrupted.Introduction of the cassette into the host cell results in insertion ofthe foreign DNA into the structural gene via the native DNA replicationmechanisms of the cell. (See for example Hamilton et al. (1989) J.Bacteriol. 171:46174622, Balbas et al. (1993) Gene 136:211-213,Gueldener et al. (1996) Nucleic Acids Res. 24:2519-2524, and Smith etal. (1996) Methods Mol. Cell. Biol. 5:270-277.)

[0156] Antisense technology is another method of down regulating geneswhere the sequence of the target gene is known. To accomplish this, anucleic acid segment from the desired gene is cloned and operably linkedto a promoter such that the anti-sense strand of RNA will betranscribed. This construct is then introduced into the host cell andthe antisense strand of RNA is produced. Antisense RNA inhibits geneexpression by preventing the accumulation of mRNA which encodes theprotein of interest. The person skilled in the art will know thatspecial considerations are associated with the use of antisensetechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of antisense genes may requirethe use of different chimeric genes utilizing different regulatoryelements known to the skilled artisan.

[0157] Although targeted gene disruption and antisense technology offereffective means of down regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence based. For example, cells may be exposed to UV radiation andthen screened for the desired phenotype. Mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect nonreplicating DNA such as HNO₂and NH₂OH, as well as agents that affect replicating DNA such asacridine dyes, notable for causing frameshift mutations. Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. See for example Thomas D. Brock in Biotechnology:A Textbook of Industrial Microbiology, Second Edition (1989) SinauerAssociates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl.Biochem. Biotechnol., 36, 227, (1992).

[0158] Another non-specific method of gene disruption is the use oftransposoable elements or transposons. Transposons are genetic elementsthat insert randomly in DNA but can be latter retrieved on the basis ofsequence to determine where the insertion has occurred. Both in vivo andin vitro transposition methods are known. Both methods involve the useof a transposable element in combination with a transposase enzyme. Whenthe transposable element or transposon, is contacted with a nucleic acidfragment in the presence of the transposase, the transposable elementwill randomly insert into the nucleic acid fragment. The technique isuseful for random mutageneis and for gene isolation, since the disruptedgene may be identified on the basis of the sequence of the transposableelement. Kits for in vitro transposition are commercially available (seefor example The Primer Island Transposition Kit, available from PerkinElmer Applied Biosystems, Branchburg, N.J., based upon the yeast Ty1element; The Genome Priming System, available from New England Biolabs,Beverly, Mass.; based upon the bacterial transposon Tn7; and the EZ::TNTransposon Insertion Systems, available from Epicentre Technologies,Madison, Wis., based upon the Tn5 bacterial transposable element.

[0159] Within the context of the present invention it may be useful tomodulate the expression of the identified isoprenoid pathway by any oneof the above described methods. For example, the present inventionprovides a number of genes encoding key enzymes in the terpenoid pathwayleading to the production of pigments and smaller isoprenoid compounds.The isolated genes include the dxs and dsr genes, the ispA, D, E, F, andG genes, the pyrG gene and the crtN genes. In particular it may beuseful to up-regulate the initial condensation of 3-carbon compounds(pyruvate and C1 aldehyde group, D-glyceraldehyde 3-Phosphate), to yieldthe 5-carbon compound D-1-deoxyxylulose-5-phosphate mediated by the dxsgene. Alternatively, if it is desired to produce a specificnon-pigmented isoprenoid, it may be desirable to disrupt various genesat the downstream end of the pathway. For example, the crtN gene isknown to encode diapophytoene dehydrogenase, which is a part of thecarotenoid biosynthesis pathway. It may be desirable to use genedisruption or antisense inhibition of this gene if a smaller, upstreamterpenoid is the desired product of the pathway.

[0160] Industrial Production

[0161] Where commercial production of the instant proteins are desired avariety of culture methodologies may be applied. For example,large-scale production of a specific gene product, overexpressed from arecombinant microbial host may be produced by both batch or continuousculture methodologies.

[0162] A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process the media is inoculated with thedesired organism or organisms and growth or metabolic activity ispermitted to occur adding nothing to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon sourceand attempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

[0163] A variation on the standard batch system is the Fed-Batch system.Fed-Batch culture processes are also suitable in the present inventionand comprise a typical batch system with the exception that thesubstrate is added in increments as the culture progresses. Fed-Batchsystems are useful when catabolite repression is apt to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the media. Measurement of the actual substrateconcentration in Fed-Batch systems is difficult and is thereforeestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases such as CO₂.Batch and Fed-Batch culturing methods are common and well known in theart and examples may be found in Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, Second Edition (1989) SinauerAssociates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl.Biochem. Biotechnol., 36, 227, (1992), herein incorporated by reference.

[0164] Commercial production of the instant proteins may also beaccomplished with a continuous culture. Continuous cultures are an opensystem where a defined culture media is added continuously to abioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high liquid phase density where cells areprimarily in log phase growth. Alternatively continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added, and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

[0165] Continuous or semi-continuous culture allows for the modulationof one factor or any number of factors that affect cell growth or endproduct concentration. For example, one method will maintain a limitingnutrient such as the carbon source or nitrogen level at a fixed rate andallow all other parameters to moderate. In other systems a number offactors affecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

[0166] Fermentation media in the present invention must contain suitablecarbon substrates. Suitable substrates may include but are not limitedto monosaccharides such as glucose and fructose, oligosaccharides suchas lactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, methane or methanol for whichmetabolic conversion into key biochemical intermediates has beendemonstrated. In addition to one and two carbon substrates,methylotrophic organisms are also known to utilize a number of othercarbon containing compounds such as methylamine, glucosamine and avariety of amino acids for metabolic activity. For example,methylotrophic yeast are known to utilize the carbon from methylamine toform trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd.,[Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly,Don P. Publisher: Intercept, Andover, UK). Similarly, various species ofCandida will metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153:485-489 (1990)). Hence it is contemplated that the sourceof carbon utilized in the present invention may encompass a wide varietyof carbon containing substrates and will only be limited by the choiceof organism.

[0167] Recombinant Expression—Plants

[0168] Plants and algae are also known to produce isoprenoid compounds.The nucleic acid fragments of the instant invention may be used tocreate transgenic plants having the ability to express the microbialprotein. Preferred plant hosts will be any variety that will support ahigh production level of the instant proteins. Suitable green plantswill included but are not limited to soybean, rapeseed (Brassica napus,B. campestris), sunflower (Helianthus annus), cotton (Gossypiumhirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativa),wheat (Triticum sp), barley (Hordeum vulgare), oats (Avena sativa, L),sorghum (Sorghum bicolor), rice (Oryza sativa), Arabidopsis, cruciferousvegetables (broccoli, cauliflower, cabbage, parsnips, etc.), melons,carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts,grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye,flax, hardwood trees, softwood trees, and forage grasses. Algal speciesinclude but not limited to commercially significant hosts such asSpirulina and Dunalliela. Overexpression of the isoprenoid compounds maybe accomplished by first constructing chimeric genes of presentinvention in which the coding region are operably linked to promoterscapable of directing expression of a gene in the desired tissues at thedesired stage of development. For reasons of convenience, the chimericgenes may comprise promoter sequences and translation leader sequencesderived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals must also be provided. The instantchimeric genes may also comprise one or more introns in order tofacilitate gene expression.

[0169] Any combination of any promoter and any terminator capable ofinducing expression of a coding region may be used in the chimericgenetic sequence. Some suitable examples of promoters and terminatorsinclude those from nopaline synthase (nos), octopine synthase (ocs) andcauliflower mosaic virus (CaMV) genes. One type of efficient plantpromoter that may be used is a high level plant promoter. Suchpromoters, in operable linkage with the genetic sequences or the presentinvention should be capable of promoting expression of the present geneproduct. High level plant promoters that may be used in this inventioninclude the promoter of the small subunit (ss) of theribulose-1,5-bisphosphate carboxylase from example from soybean(Berry-Lowe et al., J. Molecular and App. Gen., 1:483-498 1982)), andthe promoter of the chlorophyll a/b binding protein. These two promotersare known to be light-induced in plant cells (see, for example, GeneticEngineerinq of Plants, an Agricultural Perspective, A. Cashmore, Plenum,NY (1983), pages 29-38; Coruzzi, G. et al., The Journal of BiologicalChemistry, 258:1399 (1983), and Dunsmuir, P. et al., Journal ofMolecular and Applied Genetics, 2:285 (1983)).

[0170] Plasmid vectors comprising the instant chimeric genes can then beconstructed. The choice of plasmid vector depends upon the method thatwill be used to transform host plants. The skilled artisan is well awareof the genetic elements that must be present on the plasmid vector inorder to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA blots (Southern, J. Mol.Biol. 98, 503, (1975)). Northern analysis of mRNA expression (Kroczek,J. Chromatogr. Biomed. Appl., 618 (1−2) (1993)133-145), Western analysisof protein expression, or phenotypic analysis.

[0171] For some applications it will be useful to direct the instantproteins to different cellular compartments. It is thus envisioned thatthe chimeric genes described above may be further supplemented byaltering the coding sequences to encode enzymes with appropriateintracellular targeting sequences such as transit sequences (Keegstra,K., Cell 56:247-253 (1989)), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels, J. J., Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53 (1991)), or nuclear localization signals(Raikhel, N. Plant Phys. 100:1627-1632 (1992)) added and/or withtargeting sequences that are already present removed. While thereferences cited give examples of each of these, the list is notexhaustive and more targeting signals of utility may be discovered inthe future that are useful in the invention.

[0172] Protein Engineering

[0173] It is contemplated that the present nucleotides may be used toproduce gene products having enhanced or altered activity. Variousmethods are known for mutating a native gene sequence to produce a geneproduct with altered or enhanced activity including but not limited toerror prone PCR (Melnikov et al., Nucleic Acids Research, (Feb. 15,1999) Vol. 27, No. 4, pp. 1056-1062); site directed mutagenesis (Coombset al., Proteins (1998), 259-311, 1 plate. Editor(s): Angeletti, RuthHogue. Publisher: Academic, San Diego, Calif.) and “gene shuffling”(U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No.5,830,721; and U.S. Pat. No. 5,837,458, incorporated herein byreference).

[0174] The method of gene shuffling is particularly attractive due toits facile implementation, and high rate of mutagenesis and ease ofscreening. The process of gene shuffling involves the restrictionendonuclease cleavage of a gene of interest into fragments of specificsize in the presence of additional populations of DNA regions of bothsimilarity to or difference to the gene of interest. This pool offragments will then be denatured and reannealed to create a mutatedgene. The mutated gene is then screened for altered activity.

[0175] The instant microbial sequences of the present invention may bemutated and screened for altered or enhanced activity by this method.The sequences should be double stranded and can be of various lengthsranging form 50 bp to 10 kb. The sequences may be randomly digested intofragments ranging from about 10 bp to 1000 bp, using restrictionendonucleases well known in the art (Maniatis supra). In addition to theinstant microbial sequences, populations of fragments that arehybridizable to all or portions of the microbial sequence may be added.Similarly, a population of fragments which are not hybridizable to theinstant sequence may also be added. Typically these additional fragmentpopulations are added in about a 10 to 20 fold excess by weight ascompared to the total nucleic acid. Generally if this process isfollowed the number of different specific nucleic acid fragments in themixture will be about 100 to about 1000. The mixed population of randomnucleic acid fragments are denatured to form single-stranded nucleicacid fragments and then reannealed. Only those single-stranded nucleicacid fragments having regions of homology with other single-strandednucleic acid fragments will reanneal. The random nucleic acid fragmentsmay be denatured by heating. One skilled in the art could determine theconditions necessary to completely denature the double stranded nucleicacid. Preferably the temperature is from 80° C. to 100° C. The nucleicacid fragments may be reannealed by cooling. Preferably the temperatureis from 20° C. to 75° C. Renaturation can be accelerated by the additionof polyethylene glycol (“PEG”) or salt. A suitable salt concentrationmay range from 0 mM to 200 mM. The annealed nucleic acid fragments arethen incubated in the presence of a nucleic acid polymerase and dNTP's(i.e., dATP, dCTP, dGTP and dTTP). The nucleic acid polymerase may bethe Klenow fragment, the Taq polymerase or any other DNA polymeraseknown in the art. The polymerase may be added to the random nucleic acidfragments prior to annealing, simultaneously with annealing or afterannealing. The cycle of denaturation, renaturation and incubation in thepresence of polymerase is repeated for a desired number of times.Preferably the cycle is repeated from 2 to 50 times, more preferably thesequence is repeated from 10 to 40 times. The resulting nucleic acid isa larger double-stranded polynucleotide ranging from about 50 bp toabout 100 kb and may be screened for expression and altered activity bystandard cloning and expression protocol. (Manatis supra).

[0176] Furthermore, a hybrid protein can be assembled by fusion offunctional domains using the gene shuffling (exon shuffling) method(Nixon et al., PNAS, 94:1069-1073 (1997)). The functional domain of theinstant gene can be combined with the functional domain of other genesto create novel enzymes with desired catalytic function. A hybrid enzymemay be constructed using PCR overlap extension method and cloned intothe various expression vectors using the techniques well known to thoseskilled in art.

[0177] Gene Expression Profiling

[0178] All or portion of the nucleic acid fragments of the instantinvention may also be used as probes for gene expression monitoring andgene expression profiling. Many external changes such as changes ingrowth condition, exposure to chemicals, can cause induction orrepression of genes in the cell. The induction or repression of gene canbe used for a screening system to determine the best productioncondition for production organism. On the other hand, by amplifying ordisrupting genes, one can manipulate the production of the amount ofcellular products as well as the timeline. The genes may be monitoredfor expression and or regulation of expression by oxygen. It may bedesirable to deregulate or derepress these genes by knocking outregulatory elements or over-expressing regulatory elements in order toget the desired product or desired yield.

[0179] For example, all or a portion of the instant nucleic acidfragments may be immobilized on a nylon membrane or a glass slide. AGeneration 11 DNA spotter (Molecular Dynamics) is one of the availabletechnology to array the DNA samples onto the coated glass slides. Otherarray methods are also available and well known in the art. After thecells were grown in various growth conditions or treated with potentialcandidates, cellular RNA is purified. Fluorescent or radioactive labeledtarget cDNA can be made by reverse transcription of mRNA. The targetmixture is hybridized to the probes, washed using conditions well knownin the art. The amount of the target gene expression is quantified bythe intensity of radioactivity or fluorescence label (e.g., confocallaser microscope: Molecular Dynamics). The intensities of radioactivityor fluorescent label at the immobilized probes are measured using thetechnology well known in the art. The two color fluorescence detectionscheme (e.g., Cy3 and Cy5) has the advantage over radioactively labeledtargets of allowing rapid and simultaneous differential expressionanalysis of independent samples. In addition, the use of ratiomeasurements compensates for probe to probe variation of intensity dueto DNA concentration and hybridization efficiency. In the case offluorescence labeling, the two fluorescent images obtained with theappropriate excitation and emission filters constitute the raw data fromdifferential gene expression ratio values are calculated. The intensityof images are analyzed using the available software (e.g., Array Vision4.0: Imaging Research Inc.) well known in the art and normalized tocompensate for the differential efficiencies of labeling and detectionof the label. There are many different ways known in the art tonormalize the signals. One of the ways to normalize the signal is bycorrecting the signal against internal controls. Another way is to run aseparate array with labeled genomic driven DNA and compare the signalwith mRNA driven signals. This method also allows to measure thetranscript abundance. The array data of individual gene is examined andevaluated to determine the induction or repression of the gene under thetest condition.

[0180] Description of the Preferred Embodiments

[0181] The original environmental sample containing Methylomonas 16a wasobtained from pond sediment. The pond sediment was inoculated directlyinto a defined mineral medium under 25% methane in air. Methane was usedas the sole source of carbon and energy. Growth was followed until theoptical density at 660 nm was stable whereupon the culture wastransferred to fresh medium such that a 1:100 dilution was achieved.After 3 successive transfers with methane as the sole carbon and energysource, the culture was plated onto defined minimal medium agar andincubated under 25% methane in air.

[0182] The activity of the present genes and gene products has beenconfirmed by studies showing the increase in carotenoid production inthe source strain, Methylomonas 16a. By overexpressing genes that areearly in the isoprenoid pathway, dxr and dxs, an increase in carotenoidproduction was observed in Methylomonas 16a cells. Briefly, genes dxrand dxs were overexpressed in Methylomonas 16a by cloning them into thelow-copy, broad-host range plasmid pTJS75::lacZ:Tn5Kn (Schmidhauser andHelinsk, J. Bacteriology. Vol.164:446-455 (1985)). The method forcloning genes into the host plasmid is well known in the art. Genes wereamplified from the Methylomonas 16a genome via PCR with the followingprimers. Dxs primers Dxs: Primer for forward reaction:aaggatccgcgtattcgtactc. (contains a Bam HI site: SEQ ID NO:19) Dxs:Primer for reverse reaction: ctggatccgatctagaaataggctcgagttgtcgttcagg.(contains a Bam HI and a Xho I site: SEQ ID NO:20) Dxr primers: Forwardreaction: aaggatcctactcgagctgacatcagtgct. (contains a Bam HI and a Xho Isite: SEQ ID NO:21) Reverse reaction: gctctagatgcaaccagaatcg. (containsa Xba I site: SEQ ID NO:22)

[0183] The expected PCR product of dxs included a 323 bp sequenceupstream of the start codon and the expected PCR product of dxr included420 bp sequence upstream of the start codon in order to ensure that thenatural promoters of the genes were present. First, the dxs gene wascloned into the Bam HI site, which was located between the lacZ gene andthe Tn5Kn cassette of pTJS75::lacZ:Tn5Kn. The resulting plasmids wereisolated from E. coli transformants growing on LB with kanamycin (50μg/mL). The plasmid containing the insert in the direction of theKn-resistance gene (as confirmed by restriction analysis) was chosen forfurther cloning. The dxr gene was cloned in between dxs and the Tn5Kncassette using the Xho I and Xba I sites. The resulting plasmid is shownin FIG. 3b. The plasmid was transformed into E. coli usingelectroporation methods well known in the art. The presence of dxs anddxr in the plasmid was confirmed by restriction analysis and sequencing.

[0184] The plasmid pTJS75::dxs:dxr:lacZ:Tn5Kn was transferred from E.coli into Methylomonas 16a by triparental conjugation methods well knownin the art (Rainey et al., Mol. Gen. Genet. (1997), 256(1), 84-87). Aspontaneous rifampin (Rif)-resistant isolate of strain Methylomonas 16awas used as the recipient to speed the isolation of the methanotrophfrom contaminating E. coli following the mating. E. coli harboring thepTJS75::dxs:dxr:lacZ:Tn5Kn plasmid was the donor and E. coli harboringplasmid pRK2013 (Figurski and Helinski; Proc. Natl. Acad. Sci. U.S.A.76:1648-1652(1979)) served as the helper. Six separately isolatedkanamycin-resistant Methylomonas 16a transconjugants were isolated andused for the carotenoid content determination. The wild type stain andRif resistant derivative without plasmid were used as negative controls.Six transconjugants were tested for carotenoid concentration. During theextraction, pink coloration was observed in the supernatant. Thecellular carotenoid was analyzed spectrophotometrically. No qualitativedifferences were noticed in the spectra between negative controls andtransconjugants. There were no quantitative differences between the fournegative controls. There were no quantitative differences between thesix transconjugants. Transconjugants have approximately a 28% increasein carotenoid concentration when compared to the negative controls(Table 3). The overproduction of dxr and dxs genes in thetransconjugants is assumed to be the cause of the increase in thecarotenoid production in the transconjugants. Carotenoid produced in theMethylomonas cells were similar in structure as in the reference strainMethylobacterium rhodinum as seen in HPCL analysis of saponifiedextract.

EXAMPLES

[0185] The present invention is further defined in the followingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

[0186] General Methods

[0187] Standard recombinant DNA and molecular cloning techniques used inthe Examples are well known in the art and are described by Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989)(Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

[0188] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of bacterial cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

[0189] Manipulations of genetic sequences were accomplished using thesuite of programs available from the Genetics Computer Group Inc.(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.). Where the GCG program “Pileup” was used the gap creation defaultvalue of 12, and the gap extension default value of 4 were used. Wherethe CGC “Gap” or “Bestfit” programs were used the default gap creationpenalty of 50 and the default gap extension penalty of 3 were used.Multiple alignments were created using the FASTA program incorporatingthe Smith-Waterman algorithm (W. R. Pearson, Comput. Methods GenomeRes., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s):Suhai, Sandor. Publisher: Plenum, New York, N.Y.). In any case whereprogram parameters were not prompted for, in these or any otherprograms, default values were used.

[0190] The meaning of abbreviations is as follows: “h” means hour(s),“min” means minute(s), “sec” means second(s), “d” means day(s), “mL”means milliliters, “L” means liters.

Example 1 Isolation of Methylomonas 16a

[0191] The original environmental sample containing the isolate wasobtained from pond sediment. The pond sediment was inoculated directlyinto growth medium with ammonium as nitrogen source under 25% methane inair. Methane was the sole source of carbon and energy. Growth wasfollowed until the optical density at 660 nm was stable whereupon theculture was transferred to fresh medium such that a 1:100 dilution wasachieved. After 3 successive transfers with methane as sole carbon andenergy source the culture was plated onto growth agar with ammonium asnitrogen source and incubated under 25% methane in air. Manymethanotrophic bacterial species were isolated in this manner. However,Methylomonas 16a was selected as the organism to study due to the rapidgrowth of colonies, large colony size, ability to grow on minimal media,and pink pigmentation indicative of an active biosynthetic pathway forcarotenoids.

Example 2 Preparation of Genomic DNA for Sequencing and SequenceGeneration

[0192] Genomic DNA was isolated from Methylomonas according to standardprotocols.

[0193] Genomic DNA and library construction were prepared according topublished protocols (Friseur et al., The Minimal Gene Complement ofMycoplasma genitalium; Science 270,1995). A cell pellet was resuspendedin a solution containing 100 mM Na-EDTA pH 8.0, 10 mM tris-HCl pH 8.0,400 mM NaCl, and 50 mM MgCl₂.

[0194] Genomic DNA preparation. After resuspension, the cells weregently lysed in 10% SDS, and incubated for 30 min at 55° C. Afterincubation at room temperature, proteinase K was added to 100 μg/mL andincubated at 37° C. until the suspension was clear. DNA was extractedtwice with tris-equilibrated phenol and twice with chloroform. DNA wasprecipitated in 70% ethanol and resuspended in a solution containing 10mM tris-HCl and 1 mM Na-EDTA (TE) pH 7.5. The DNA solution was treatedwith a mix of RNAases, then extracted twice with tris-equilibratedphenol and twice with chloroform. This was followed by precipitation inethanol and resuspension in TE.

[0195] Library construction. 200 to 500 μg of chromosomal DNA wasresuspended in a solution of 300 mM sodium acetate, 10 mM tris-HCl, 1 mMNa-EDTA, and 30% glycerol, and sheared at 12 psi for 60 sec in anAeromist Downdraft Nebulizer chamber (IBI Medical products, Chicago,Ill.). The DNA was precipitated, resuspended and treated with Bal31nuclease. After size fractionation, a fraction (2.0 kb, or 5.0 kb) wasexcised, cleaned and a two-step ligation procedure was used to produce ahigh titer library with greater than 99% single inserts.

[0196] Sequencing. A shotgun sequencing strategy approach was adoptedfor the sequencing of the whole microbial genome (Fleischmann, Robert etal., Whole-Genome Random sequencing and assembly of Haemophilusinfluenzae Rd Science, 269:1995).

[0197] Sequence was generated on an ABI Automatic sequencer using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in either DNAStar (DNA Star Inc.) or the Wisconsin GCG program(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.) and the CONSED package (version 7.0). All sequences representcoverage at least two times in both directions.

EXAMPLE 3 Identification and Characterization of Bacterial ORF's

[0198] ORFs encoding 1-9 were initially identified by conducting BLAST(Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J.Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searchesfor similarity to sequences contained in the BLAST “nr” database(comprising all non-redundant (nr) GenBank CDS translations, sequencesderived from the 3-dimensional structure Brookhaven Protein Data Bank,the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). Thesequences obtained in Example 2 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTP algorithm(Altschul, S. F., et al., Nucleic Acid Res. 25:3389-3402) (1997)provided by the NCBI.

[0199] All initial comparisons were done using either the BLASTNnr orBLASTPnr algorithm. A refined similarity search was performed usingFASTA (version 3.2) with the default parameters settings (BLOSUM 50scoring matrix, word size ktup=2, gap penalty=−12 for the first residueand −2 for every additional residue in the gap). The results of theFASTA comparison is given in Table 1 which summarize the sequences towhich they have the most similarity. Table 1 displays data based on theFASTA algorithm with values reported in expect values. The Expect valueestimates the statistical significance of the match, specifying thenumber of matches, with a given score, that are expected in a search ofa database of this size absolutely by chance.

[0200] A gene cluster of ispD, ispF and pyrG and another gene cluster ofgenes crtN1 and crtN2 are shown in FIG. 2. TABLE 1 ORF Gene SEQ ID % %Name Name Similarity Identified SEQ ID peptide Identity^(a)similarity^(b) E-value^(c) Citation 1 dxs 1-deoxyxylulose-5- 1 2 60% 86% 5.7e−149 Lois et al., Proc. Natl. Acad. phosphate synthase Sci. USA. 95(5), 2105-2110 (E. coli) (1998) 2 dxr 1-deoxy-d-xylulose 5- 3 4 55% 78%3.3e−74 Takahashi et al., Proc. Natl. phosphate Acad. USA 95: 9879-9884reductoisomerase (1998) (E. coli) 3 ygbP/ispD 2C-methyl-d-erythritol 5 652% 74% 7.7e−36 Rohdich et al., Proc Natl cytidylyltransferase Acad SciUSA 1999 Oct (E. coli) 12; 96(21): 11758-63 4 ychB/IspE4-diphosphocytidyl-2-C- 7 8 50% 73% 8.8e−49 Luttgen et al., Proc Natlmethylerythritol kinase Acad Sci USA. 2000 Feb (E. coli) 1; 97(3):1062-7. 5 ygbB/ispF 2C-methyl-d-erythritol 9 10 69% 84% 1.6e−36 Herz etal., 2,4-cyclodiphosphate Proc Natl Acad Sci USA synthase 2000 Mar 14;97(6): 2486-90 (E. coli) 6 pyrG CTP synthase 11 12 67% 89%  2.4e−141Weng. et al., J. Biol. Chem. (E. coli) 261: 5568-5574 (1986) 7 IspAGeranyltranstransferase 13 14 57% 78% 7.8e−56 Ohto, et al., Plant Mol.Biol. (also farnesyl- 40 (2), 307-321 (1999) diphosphate synthase)(Synechococcus elongatus) 8 crtN1 diapophytoene 15 16 34% 72%   4e−66Xiong, et al., .″ dehydrogenase CrtN- Proc. Natl. Acad. Sci. U.S.A. copy1 95 (25), 14851-14856 (1998) (Heliobacillus mobilis) 9 crtN2Diapophytoene 17 18 49% 78% 1.3e−76 Genbank #: X97985 dehydrogenaseCrtN- copy 2 (Staphylococcus aureus) 10 lytB Acinetobacter sp BD413 2324 65  87  3.4e−75 Genbank # G.I. 5915671 Putative penicillin bindingprotein*

EXAMPLE 4 Up Regulation of dxs and dxr Genes

[0201] For the cloning, the low-copy, broad-host plasmid,pTJS75::lacZ:Tn5Kn was used (Schmidhauser and Helinski J. Bacteriology.Vol.164:446-455 (1985). Genes dxs and dxr were amplified from theMethylomonas 16a genome by using PCR with the following primers.

[0202] Dxs Primers:

[0203] Forward reaction: aaggatccgcgtattcgtactc (contains a Bam HI site,SEQ ID NO:19).

[0204] Reverse reaction: ctggatccgatctagaaataggctcgagttgtcgttcagg(contains a Bam HI and a Xho I site, SEQ ID NO:20).

[0205] Dxr Primers:

[0206] Forward reaction: aaggatcctactcgagctgacatcagtgct (contains a BamHI and a Xho I site, SEQ ID NO:21).

[0207] Reverse reaction: gctctagatgcaaccagaatcg (contains a Xba I site,SEQ ID NO:22).

[0208] The expected PCR products of dxs and dxr genes included sequencesof 323 bp and 420 bp, respectively, upstream of the start codon of eachgene in order to ensure that the natural promoters of the genes werepresent. PCR program (in Perkin-Elmer, Norwalk, CT): Activation: 95°C. - 900 sec Cycle (35 times): 94° C. - 45 sec 58° C. - 45 sec 72° C. -60 sec Final elongation: 72° C. - 600 sec

[0209] PCR Reaction mixture: 25 μl Hot Star master mix (Qiagen,Valencia, CA) 0.75 μl genomic DNA (approx. 0.1 ng) 1.2 μl sense primer(= 10 pmol) 1.2 μl antisense primer (= 10 pmol) 21.85 μl deionized water50 μl

[0210] Standard procedures (Sambrook, J., Fritsch, E. F. and Maniatis,T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor (1989)), were used in orderto clone dxs and dxr into pTJS75::lacZ:Tn5Kn:

[0211] For isolation, concentration, and purification of DNA, Qiagenkits (Valencia, Calif.) were used. Enzymes for the cloning werepurchased from Gibco/BRL (Rockville, Md.) or NEB (Beverly, Mass.). Totransfer plasmids into E. coli, One Shot Top10 competent cells(Invitrogen, Carlsbad, Calif.), cuvettes (0.2 cm; Invitrogen), andBio-Rad Gene Pulser III (Hercules, Calif.) with standard settings wereused for electroporation. TABLE 2 BTZ medium for Methylomonas 16a Conc.g per MW (mM) L Composition: NaNO₃ 84.99 10  0.85 KH₂PO₄ 136.09 3.67 0.5Na₂SO₄ 142.04 3.52 0.5 MgCl₂ × 6H₂O 203.3 0.98 0.2 CaCl₂ × 2H₂O 147.020.68 0.1 1 M HEPES (pH 238.3 50 mL 7) Solution 1 10 mL Solution 1 (metalsolution) Nitriloacetic acid 191.1 66.9 12.8 CuCl₂ × 2H₂O 170.48 0.150.0254 FeCl₂ × 4H₂O 198.81 1.5 0.3 MnCl₂ × 4H₂O 197.91 0.5 0.1 CoCl₂ ×6H₂O 237.9 1.31 0.312 ZnCl₂ 136.29 0.73 0.1 H₃BO₃ 61.83 0.16 0.01Na₂MoO₄ × 241.95 0.04 0.01 2H₂O NiCl₂ × 6H₂O 237.7 0.77 0.184

[0212] First, dxs was cloned into the Bam HI site, which was locatedbetween the lacZ gene and the Tn5Kn cassette of pTJS75::lacZ:Tn5Kn. Theresulting plasmids were isolated from E. coli transformants growing onLB+kanamycin (Kn, 50 μg/mL). The plasmid containing the insert indirection of the Kn-resistance gene (as confirmed by restrictionanalysis) was chosen for further cloning. Dxr gene was cloned in betweendxs and Tn5Kn cassette by using the Xho I and Xba I sites. Theanticipated plasmid was isolated from E. coli transformants. Thepresence of dxs and dxr in the plasmid was confirmed by restrictionanalysis and sequencing. The resulting plasmid,pTJS75::dxs:dxr:lacZ:Tn5Kn is shown in FIG. 3b.

[0213] 16a Transconiugants

[0214] The plasmid pTJS75::dxs:dxr:lacZ:Tn5Kn was transferred from E.coli into Methylomonas 16a by triparental conjugation well known in theart (Rainey et al., Mol. Gen. Genet. (1997), 256(1), 84-87).

[0215] A spontaneous rifampin (Rif)-resistant isolate of strainMethylomonas 16a was used as the recipient to speed the isolation of themethanotroph from contaminating E. coli following the mating. E. coliharboring the pTJS75::dxs:dxr:lacZ:Tn5Kn plasmid was the donor and E.coli harboring plasmid pRK2013 (Figurski and Helinski; Proc. Natl. Acad.Sci. U.S.A. 76:1648-1652(1979)) served as the helper. The approximaterelative cell concentrations on the plates wererecipient:donor:helper=2:1:1.

[0216] The corresponding LB plates were incubated under methane (25%) at30° C. overnight. Then the mating mixtures were scraped off the plates,resuspended in 1 mL of BTZ medium (Table 2), and plated onto BTZ platessupplemented with Rif (25 μg/mL) and Kn (50 μg/mL). The plates wereincubated under methane (25%) for 7 days at 30° C. to select forMethylomonas 16a transconjugants. Upcoming colonies were picked andtransferred to fresh selection plates for further purification. Sixseparately isolated kanamycin-resistant Methylomonas 16a transconjugantswere used for carotenoid content determination.

[0217] For carotenoid determination, six 100 mL cultures oftransconjugants (in BTZ+50 μg/mL Kn) were grown under methane (25%) overthe weekend to stationary growth phase. Two cultures of each, thewild-type strain and its Rif-resistant derivative without the plasmid,served as a control to see whether there are different carotenoidcontents in those strains and to get a standard deviation of thecarotenoid determination. Cells were spun down, washed with distilledwater, and freeze-dried (lyophilizer: Virtis, Gardiner, N.Y.) for 24 hin order to determine dry-weights. After the dry-weight of each culture,was determined, cells were extracted. First, cells were welled with 0.4mL of water and let stand for 15 min. After 15 min, four mL of acetonewas added and thoroughly vortexed to homogenize the sample. The sampleswere then shaken at 30° C. for 1 hr. After 1 hr, the cells werecentrifuged. Pink coloration was observed in the supernatant. Thesupernatant was collected and pellets were extracted again with 0.3 mLof water and 3 mL of acetone. The supernatants from the secondextraction were lighter pink in color. The supernatants of bothextractions were combined, their volumes were measured, and analyzedspectrophotometrically. No qualitative differences were seen in thespectra between negative control and transconjugant samples. In acetoneextract, a following observation was typical measured byspectrophotometer (shoulder at 460 nm, maxima at 491 and 522 nm)(Amersham Pharmacia Biotech, Piscataway, N.J.). For calculation of thecarotenoid content, the absorption at 491 nm was read, the molarextinction coefficient of bacterioruberin (188,000) and a MW of 552 wereused. The MW of the carotenoid (552 g/mol) was determined by MALDI-MS ofa purified sample (Silica/Mg adsorption followed by Silica columnchromatography, reference: Britton, G., Liaaen-Jensen, S., Pfander, H.,Carotenoids Vol. 1a; Isolation and analysis, Birkhauser Verlag, Basel,Boston, Berlin (1995)).

[0218] A crude acetone extract from Methylomonas 16a cells has a typicalabsorption spectrum (inflexion at 460 nm, maxima at 491 nm and 522 nm).HPLC analysis (Beckman Gold Nouveau System, Columbia, Md.; Conditions:125×4 mm RP8 (5 μm particles) column with corresponding guard column(Hewlett-Packard, San Fernando, Calif.); flow 1 mL/min; solvent program:0-10 min 15% water/85% methanol, then 100% methanol) of acetone extractsconfirmed that one major carotenoid (net retention volume at about 6 mL)with above mentioned absorption spectrum is responsible for the pinkcoloration of wild-type and transconjugant Methylomonas 16a cells.Because nothing else in the extract absorbs at 491 nm, carotenoidcontent was directly measured in the acetone extract with aspectrophotometer (Amersham Pharmacia Biotech, Piscataway, N.J.).

[0219] The molar extinction coefficient of bacterioruberin (188,000),was used for the calculation of the quantity.

[0220] The following formula was used (Lambert-Beer's law) to determinethe quantity of carotenoid:

[0221] Ca=A_(491 nm)/(d×ε×v×MW)

[0222] Ca: Carotenoid amount (g)

[0223] A_(491 nm): Absorption of acetone extract at 491 nm (−)

[0224] d: Light path in cuvette (1 cm)

[0225] ε: Molar extinction coefficient (U(mol×cm))

[0226] MW: Molecular weight (g/mol)

[0227] v: Volume of extract (L)

[0228] To get the carotenoid content, the calculated carotenoid amounthas to be divided by the corresponding cell dry weight. TABLE 3Carotenoid contents in Methylomonas 16a cells dry weight carotenoidcontent Cultures (mg) carotenoid (g) (μg/g) 16a-1^(a) 30.8 3.00194E − 06 97.5 16a-2^(a) 30.8 3.0865E − 06 100.2 16a Rif-1^(b) 29.2 3.12937E − 06107.2 16a Rif-2^(b) 30.1 3.02014E − 06 100.3 dxp 1^(c) 28.2 3.48817E −06 123.7 dxp 2^(c) 23.8 3.17224E − 06 133.3 dxp 3^(c) 31.6 4.01962E − 06127.2 dxp 4^(c) 31.8 4.38899E − 06 138.0 dxp 5^(c) 28.4 3.4547E − 06121.6 dxp 6^(c) 30.3 4.00817E − 06 132.3

[0229] There were no significant differences between four negativecontrols. Likewise, there were no significant differences between sixtransconjugants. However, approximately 28% increase in averagecarotenoid production was observed in the transconjugants in comparisonto the average carotenoid production in negative controls (Table 3).

[0230] In order to confirm the structure, Methylobacterium rhodinum(formerly Pseudomonas rhodos: ATCC No.14821) of which C30-carotenoid wasidentified was used as a reference strain (Keinig et al., Z. Naturforsch34c, 181-185 (1979); Kleinig and Schmitt, Z. Naturforsch 37c, 758-760(1982)). A saponified extract of Methylobacterium rhodinum and ofMethylomonas 16a were compared by HPLC analysis under the sameconditions as mentioned above. The results are shown as follows:Saponified M. rhodinum: inflexion at 460 nm, maxima at 487 nm, 517 nm.

[0231] Net retention volume=1.9 mL.

[0232] Saponified Methylomonas 16a: inflexion at 460 nm, maxima at 488nm, 518 nm.

[0233] Net retention volume=2.0 mL.

1 24 1 1860 DNA Methylomonas 16a ORF1 1 atgaaactga ccaccgacta tcccttgcttaaaaacatcc acacgccggc ggacatacgc 60 gcgctgtcca aggaccagct ccagcaactggctgacgagg tgcgcggcta tctgacccac 120 acggtcagca tttccggcgg ccattttgcggccggcctcg gcaccgtgga actgaccgtg 180 gccttgcatt atgtgttcaa tacccccgtcgatcagttgg tctgggacgt gggccatcag 240 gcctatccgc acaagattct gaccggtcgcaaggagcgca tgccgaccat tcgcaccctg 300 ggcggggtgt cagcctttcc ggcgcgggacgagagcgaat acgatgcctt cggcgtcggc 360 cattccagca cctcgatcag cgcggcactgggcatggcca ttgcgtcgca gctgcgcggc 420 gaagacaaga agatggtagc catcatcggcgacggttcca tcaccggcgg catggcctat 480 gaggcgatga atcatgccgg cgatgtgaatgccaacctgc tggtgatctt gaacgacaac 540 gatatgtcga tctcgccgcc ggtcggggcgatgaacaatt atctgaccaa ggtgttgtcg 600 agcaagtttt attcgtcggt gcgggaagagagcaagaaag ctctggccaa gatgccgtcg 660 gtgtgggaac tggcgcgcaa gaccgaggaacacgtgaagg gcatgatcgt gcccggtacc 720 ttgttcgagg aattgggctt caattatttcggcccgatcg acggccatga tgtcgagatg 780 ctggtgtcga ccctggaaaa tctgaaggatttgaccgggc cggtattcct gcatgtggtg 840 accaagaagg gcaaaggcta tgcgccagccgagaaagacc cgttggccta ccatggcgtg 900 ccggctttcg atccgaccaa ggatttcctgcccaaggcgg cgccgtcgcc gcatccgacc 960 tataccgagg tgttcggccg ctggctgtgcgacatggcgg ctcaagacga gcgcttgctg 1020 ggcatcacgc cggcgatgcg cgaaggctctggtttggtgg aattctcaca gaaatttccg 1080 aatcgctatt tcgatgtcgc catcgccgagcagcatgcgg tgaccttggc cgccggccag 1140 gcctgccagg gcgccaagcc ggtggtggcgatttattcca ccttcctgca acgcggttac 1200 gatcagttga tccacgacgt ggccttgcagaacttagata tgctctttgc actggatcgt 1260 gccggcttgg tcggcccgga tggaccgacccatgctggcg cctttgatta cagctacatg 1320 cgctgtattc cgaacatgct gatcatggctccagccgacg agaacgagtg caggcagatg 1380 ctgaccaccg gcttccaaca ccatggcccggcttcggtgc gctatccgcg cggcaaaggg 1440 cccggggcgg caatcgatcc gaccctgaccgcgctggaga tcggcaaggc cgaagtcaga 1500 caccacggca gccgcatcgc cattctggcctggggcagca tggtcacgcc tgccgtcgaa 1560 gccggcaagc agctgggcgc gacggtggtgaacatgcgtt tcgtcaagcc gttcgatcaa 1620 gccttggtgc tggaattggc caggacgcacgatgtgttcg tcaccgtcga ggaaaacgtc 1680 atcgccggcg gcgctggcag tgcgatcaacaccttcctgc aggcgcagaa ggtgctgatg 1740 ccggtctgca acatcggcct gcccgaccgcttcgtcgagc aaggtagtcg cgaggaattg 1800 ctcagcctgg tcggcctcga cagcaagggcatcctcgcca ccatcgaaca gttttgcgct 1860 2 620 PRT Methylomonas 16a Aminoacid sequences encoded by ORF1 2 Met Lys Leu Thr Thr Asp Tyr Pro Leu LeuLys Asn Ile His Thr Pro 1 5 10 15 Ala Asp Ile Arg Ala Leu Ser Lys AspGln Leu Gln Gln Leu Ala Asp 20 25 30 Glu Val Arg Gly Tyr Leu Thr His ThrVal Ser Ile Ser Gly Gly His 35 40 45 Phe Ala Ala Gly Leu Gly Thr Val GluLeu Thr Val Ala Leu His Tyr 50 55 60 Val Phe Asn Thr Pro Val Asp Gln LeuVal Trp Asp Val Gly His Gln 65 70 75 80 Ala Tyr Pro His Lys Ile Leu ThrGly Arg Lys Glu Arg Met Pro Thr 85 90 95 Ile Arg Thr Leu Gly Gly Val SerAla Phe Pro Ala Arg Asp Glu Ser 100 105 110 Glu Tyr Asp Ala Phe Gly ValGly His Ser Ser Thr Ser Ile Ser Ala 115 120 125 Ala Leu Gly Met Ala IleAla Ser Gln Leu Arg Gly Glu Asp Lys Lys 130 135 140 Met Val Ala Ile IleGly Asp Gly Ser Ile Thr Gly Gly Met Ala Tyr 145 150 155 160 Glu Ala MetAsn His Ala Gly Asp Val Asn Ala Asn Leu Leu Val Ile 165 170 175 Leu AsnAsp Asn Asp Met Ser Ile Ser Pro Pro Val Gly Ala Met Asn 180 185 190 AsnTyr Leu Thr Lys Val Leu Ser Ser Lys Phe Tyr Ser Ser Val Arg 195 200 205Glu Glu Ser Lys Lys Ala Leu Ala Lys Met Pro Ser Val Trp Glu Leu 210 215220 Ala Arg Lys Thr Glu Glu His Val Lys Gly Met Ile Val Pro Gly Thr 225230 235 240 Leu Phe Glu Glu Leu Gly Phe Asn Tyr Phe Gly Pro Ile Asp GlyHis 245 250 255 Asp Val Glu Met Leu Val Ser Thr Leu Glu Asn Leu Lys AspLeu Thr 260 265 270 Gly Pro Val Phe Leu His Val Val Thr Lys Lys Gly LysGly Tyr Ala 275 280 285 Pro Ala Glu Lys Asp Pro Leu Ala Tyr His Gly ValPro Ala Phe Asp 290 295 300 Pro Thr Lys Asp Phe Leu Pro Lys Ala Ala ProSer Pro His Pro Thr 305 310 315 320 Tyr Thr Glu Val Phe Gly Arg Trp LeuCys Asp Met Ala Ala Gln Asp 325 330 335 Glu Arg Leu Leu Gly Ile Thr ProAla Met Arg Glu Gly Ser Gly Leu 340 345 350 Val Glu Phe Ser Gln Lys PhePro Asn Arg Tyr Phe Asp Val Ala Ile 355 360 365 Ala Glu Gln His Ala ValThr Leu Ala Ala Gly Gln Ala Cys Gln Gly 370 375 380 Ala Lys Pro Val ValAla Ile Tyr Ser Thr Phe Leu Gln Arg Gly Tyr 385 390 395 400 Asp Gln LeuIle His Asp Val Ala Leu Gln Asn Leu Asp Met Leu Phe 405 410 415 Ala LeuAsp Arg Ala Gly Leu Val Gly Pro Asp Gly Pro Thr His Ala 420 425 430 GlyAla Phe Asp Tyr Ser Tyr Met Arg Cys Ile Pro Asn Met Leu Ile 435 440 445Met Ala Pro Ala Asp Glu Asn Glu Cys Arg Gln Met Leu Thr Thr Gly 450 455460 Phe Gln His His Gly Pro Ala Ser Val Arg Tyr Pro Arg Gly Lys Gly 465470 475 480 Pro Gly Ala Ala Ile Asp Pro Thr Leu Thr Ala Leu Glu Ile GlyLys 485 490 495 Ala Glu Val Arg His His Gly Ser Arg Ile Ala Ile Leu AlaTrp Gly 500 505 510 Ser Met Val Thr Pro Ala Val Glu Ala Gly Lys Gln LeuGly Ala Thr 515 520 525 Val Val Asn Met Arg Phe Val Lys Pro Phe Asp GlnAla Leu Val Leu 530 535 540 Glu Leu Ala Arg Thr His Asp Val Phe Val ThrVal Glu Glu Asn Val 545 550 555 560 Ile Ala Gly Gly Ala Gly Ser Ala IleAsn Thr Phe Leu Gln Ala Gln 565 570 575 Lys Val Leu Met Pro Val Cys AsnIle Gly Leu Pro Asp Arg Phe Val 580 585 590 Glu Gln Gly Ser Arg Glu GluLeu Leu Ser Leu Val Gly Leu Asp Ser 595 600 605 Lys Gly Ile Leu Ala ThrIle Glu Gln Phe Cys Ala 610 615 620 3 1182 DNA Methylomonas 16a ORF2 3atgaaaggta tttgcatatt gggcgctacc ggttcgatcg gtgtcagcac gctggatgtc 60gttgccaggc atccggataa atatcaagtc gttgcgctga ccgccaacgg caatatcgac 120gcattgtatg aacaatgcct ggcccaccat ccggagtatg cggtggtggt catggaaagc 180aaggtagcag agttcaaaca gcgcattgcc gcttcgccgg tagcggatat caaggtcttg 240tcgggtagcg aggccttgca acaggtggcc acgctggaaa acgtcgatac ggtgatggcg 300gctatcgtcg gcgcggccgg attgttgccg accttggccg cggccaaggc cggcaaaacc 360gtgctgttgg ccaacaagga agccttggtg atgtcgggac aaatcttcat gcaggccgtc 420agcgattccg gcgctgtgtt gctgccgata gacagcgagc acaacgccat ctttcagtgc 480atgccggcgg gttatacgcc aggccataca gccaaacagg cgcgccgcat tttattgacc 540gcttccggtg gcccatttcg acggacgccg atagaaacgt tgtccagcgt cacgccggat 600caggccgttg cccatcctaa atgggacatg gggcgcaaga tttcggtcga ttccgccacc 660atgatgaaca aaggtctcga actgatcgaa gcctgcttgt tgttcaacat ggagcccgac 720cagattgaag tcgtcattca tccgcagagc atcattcatt cgatggtgga ctatgtcgat 780ggttcggttt tggcgcagat gggtaatccc gacatgcgca cgccgatagc gcacgcgatg 840gcctggccgg aacgctttga ctctggtgtg gcgccgctgg atattttcga agtagggcac 900atggatttcg aaaaacccga cttgaaacgg tttccttgtc tgagattggc ttatgaagcc 960atcaagtctg gtggaattat gccaacggta ttgaacgcag ccaatgaaat tgctgtcgaa 1020gcgtttttaa atgaagaagt caaattcact gacatcgcgg tcatcatcga gcgcagcatg 1080gcccagttta aaccggacga tgccggcagc ctcgaattgg ttttgcaggc cgatcaagat 1140gcgcgcgagg tggctagaga catcatcaag accttggtag ct 1182 4 394 PRTMethylomonas 16a Amino acid sequences encoded by ORF2 4 Met Lys Gly IleCys Ile Leu Gly Ala Thr Gly Ser Ile Gly Val Ser 1 5 10 15 Thr Leu AspVal Val Ala Arg His Pro Asp Lys Tyr Gln Val Val Ala 20 25 30 Leu Thr AlaAsn Gly Asn Ile Asp Ala Leu Tyr Glu Gln Cys Leu Ala 35 40 45 His His ProGlu Tyr Ala Val Val Val Met Glu Ser Lys Val Ala Glu 50 55 60 Phe Lys GlnArg Ile Ala Ala Ser Pro Val Ala Asp Ile Lys Val Leu 65 70 75 80 Ser GlySer Glu Ala Leu Gln Gln Val Ala Thr Leu Glu Asn Val Asp 85 90 95 Thr ValMet Ala Ala Ile Val Gly Ala Ala Gly Leu Leu Pro Thr Leu 100 105 110 AlaAla Ala Lys Ala Gly Lys Thr Val Leu Leu Ala Asn Lys Glu Ala 115 120 125Leu Val Met Ser Gly Gln Ile Phe Met Gln Ala Val Ser Asp Ser Gly 130 135140 Ala Val Leu Leu Pro Ile Asp Ser Glu His Asn Ala Ile Phe Gln Cys 145150 155 160 Met Pro Ala Gly Tyr Thr Pro Gly His Thr Ala Lys Gln Ala ArgArg 165 170 175 Ile Leu Leu Thr Ala Ser Gly Gly Pro Phe Arg Arg Thr ProIle Glu 180 185 190 Thr Leu Ser Ser Val Thr Pro Asp Gln Ala Val Ala HisPro Lys Trp 195 200 205 Asp Met Gly Arg Lys Ile Ser Val Asp Ser Ala ThrMet Met Asn Lys 210 215 220 Gly Leu Glu Leu Ile Glu Ala Cys Leu Leu PheAsn Met Glu Pro Asp 225 230 235 240 Gln Ile Glu Val Val Ile His Pro GlnSer Ile Ile His Ser Met Val 245 250 255 Asp Tyr Val Asp Gly Ser Val LeuAla Gln Met Gly Asn Pro Asp Met 260 265 270 Arg Thr Pro Ile Ala His AlaMet Ala Trp Pro Glu Arg Phe Asp Ser 275 280 285 Gly Val Ala Pro Leu AspIle Phe Glu Val Gly His Met Asp Phe Glu 290 295 300 Lys Pro Asp Leu LysArg Phe Pro Cys Leu Arg Leu Ala Tyr Glu Ala 305 310 315 320 Ile Lys SerGly Gly Ile Met Pro Thr Val Leu Asn Ala Ala Asn Glu 325 330 335 Ile AlaVal Glu Ala Phe Leu Asn Glu Glu Val Lys Phe Thr Asp Ile 340 345 350 AlaVal Ile Ile Glu Arg Ser Met Ala Gln Phe Lys Pro Asp Asp Ala 355 360 365Gly Ser Leu Glu Leu Val Leu Gln Ala Asp Gln Asp Ala Arg Glu Val 370 375380 Ala Arg Asp Ile Ile Lys Thr Leu Val Ala 385 390 5 693 DNAMethylomonas 16a ORF3 5 atgaacccaa ccatccaatg ctgggccgtc gtgcccgcagccggcgtcgg caaacgcatg 60 caagccgatc gccccaaaca atatttaccg cttgccggtaaaacggtcat cgaacacaca 120 ctgactcgac tacttgagtc cgacgccttc caaaaagttgcggtggcgat ttccgtcgaa 180 gacccttatt ggcctgaact gtccatagcc aaacaccccgacatcatcac cgcgcctggc 240 ggcaaggaac gcgccgactc ggtgctgtct gcactgaaggctttagaaga tatagccagc 300 gaaaatgatt gggtgctggt acacgacgcc gcccgcccctgcttgacggg cagcgacatc 360 caccttcaaa tcgatacctt aaaaaatgac ccggtcggcggcatcctggc cttgagttcg 420 cacgacacat tgaaacacgt ggatggtgac acgatcaccgcaaccataga cagaaagcac 480 gtctggcgcg ccttgacgcc gcaaatgttc aaatacggcatgttgcgcga cgcgttgcaa 540 cgaaccgaag gcaatccggc cgtcaccgac gaagccagtgcgctggaact tttgggccat 600 aaacccaaaa tcgtggaagg ccgcccggac aacatcaaaatcacccgccc ggaagatttg 660 gccctggcac aattttatat ggagcaacaa gca 693 6 231PRT Methylomonas 16a Amino acid sequences encoded by ORF3 6 Met Asn ProThr Ile Gln Cys Trp Ala Val Val Pro Ala Ala Gly Val 1 5 10 15 Gly LysArg Met Gln Ala Asp Arg Pro Lys Gln Tyr Leu Pro Leu Ala 20 25 30 Gly LysThr Val Ile Glu His Thr Leu Thr Arg Leu Leu Glu Ser Asp 35 40 45 Ala PheGln Lys Val Ala Val Ala Ile Ser Val Glu Asp Pro Tyr Trp 50 55 60 Pro GluLeu Ser Ile Ala Lys His Pro Asp Ile Ile Thr Ala Pro Gly 65 70 75 80 GlyLys Glu Arg Ala Asp Ser Val Leu Ser Ala Leu Lys Ala Leu Glu 85 90 95 AspIle Ala Ser Glu Asn Asp Trp Val Leu Val His Asp Ala Ala Arg 100 105 110Pro Cys Leu Thr Gly Ser Asp Ile His Leu Gln Ile Asp Thr Leu Lys 115 120125 Asn Asp Pro Val Gly Gly Ile Leu Ala Leu Ser Ser His Asp Thr Leu 130135 140 Lys His Val Asp Gly Asp Thr Ile Thr Ala Thr Ile Asp Arg Lys His145 150 155 160 Val Trp Arg Ala Leu Thr Pro Gln Met Phe Lys Tyr Gly MetLeu Arg 165 170 175 Asp Ala Leu Gln Arg Thr Glu Gly Asn Pro Ala Val ThrAsp Glu Ala 180 185 190 Ser Ala Leu Glu Leu Leu Gly His Lys Pro Lys IleVal Glu Gly Arg 195 200 205 Pro Asp Asn Ile Lys Ile Thr Arg Pro Glu AspLeu Ala Leu Ala Gln 210 215 220 Phe Tyr Met Glu Gln Gln Ala 225 230 7855 DNA Methylomonas 16a ORF4 7 atggattatg cggctgggtg gggcgaaagatggcctgctc cggcaaaatt gaacttaatg 60 ttgaggatta ccggtcgcag gccagatggctatcatctgt tgcaaacggt gtttcaaatg 120 ctcgatctat gcgattggtt gacgtttcatccggttgatg atggccgcgt gacgctgcga 180 aatccaatct ccggcgttcc agagcaggatgacttgactg ttcgggcggc taatttgttg 240 aagtctcata ccggctgtgt gcgcggagtttgtatcgata tcgagaaaaa tctgcctatg 300 ggtggtggtt tgggtggtgg aagttccgatgctgctacaa ccttggtagt tctaaatcgg 360 ctttggggct tgggcttgtc gaagcgtgagttgatggatt tgggcttgag gcttggtgcc 420 gatgtgcctg tgtttgtgtt tggttgttcggcctggggcg aaggtgtgag cgaggatttg 480 caggcaataa cgttgccgga acaatggtttgtcatcatta aaccggattg ccatgtgaat 540 actggagaaa ttttttctgc agaaaatttgacaaggaata gtgcagtcgt tacaatgagc 600 gactttcttg caggggataa tcggaatgattgttcggaag tggtttgcaa gttatatcga 660 ccggtgaaag atgcaatcga tgcgttgttatgctatgcgg aagcgagatt gacggggacc 720 ggtgcatgtg tgttcgctca gttttgtaacaaggaagatg ctgagagtgc gttagaagga 780 ttgaaagatc ggtggctggt gttcttggctaaaggcttga atcagtctgc gctctacaag 840 aaattagaac aggga 855 8 285 PRTMethylomonas 16a Amino acid sequences encoded by ORF4 8 Met Asp Tyr AlaAla Gly Trp Gly Glu Arg Trp Pro Ala Pro Ala Lys 1 5 10 15 Leu Asn LeuMet Leu Arg Ile Thr Gly Arg Arg Pro Asp Gly Tyr His 20 25 30 Leu Leu GlnThr Val Phe Gln Met Leu Asp Leu Cys Asp Trp Leu Thr 35 40 45 Phe His ProVal Asp Asp Gly Arg Val Thr Leu Arg Asn Pro Ile Ser 50 55 60 Gly Val ProGlu Gln Asp Asp Leu Thr Val Arg Ala Ala Asn Leu Leu 65 70 75 80 Lys SerHis Thr Gly Cys Val Arg Gly Val Cys Ile Asp Ile Glu Lys 85 90 95 Asn LeuPro Met Gly Gly Gly Leu Gly Gly Gly Ser Ser Asp Ala Ala 100 105 110 ThrThr Leu Val Val Leu Asn Arg Leu Trp Gly Leu Gly Leu Ser Lys 115 120 125Arg Glu Leu Met Asp Leu Gly Leu Arg Leu Gly Ala Asp Val Pro Val 130 135140 Phe Val Phe Gly Cys Ser Ala Trp Gly Glu Gly Val Ser Glu Asp Leu 145150 155 160 Gln Ala Ile Thr Leu Pro Glu Gln Trp Phe Val Ile Ile Lys ProAsp 165 170 175 Cys His Val Asn Thr Gly Glu Ile Phe Ser Ala Glu Asn LeuThr Arg 180 185 190 Asn Ser Ala Val Val Thr Met Ser Asp Phe Leu Ala GlyAsp Asn Arg 195 200 205 Asn Asp Cys Ser Glu Val Val Cys Lys Leu Tyr ArgPro Val Lys Asp 210 215 220 Ala Ile Asp Ala Leu Leu Cys Tyr Ala Glu AlaArg Leu Thr Gly Thr 225 230 235 240 Gly Ala Cys Val Phe Ala Gln Phe CysAsn Lys Glu Asp Ala Glu Ser 245 250 255 Ala Leu Glu Gly Leu Lys Asp ArgTrp Leu Val Phe Leu Ala Lys Gly 260 265 270 Leu Asn Gln Ser Ala Leu TyrLys Lys Leu Glu Gln Gly 275 280 285 9 471 DNA Methylomonas 16a ORF5 9atgatacgcg taggcatggg ttacgacgtg caccgtttca acgacggcga ccacatcatt 60ttgggcggcg tcaaaatccc ttatgaaaaa ggcctggaag cccattccga cggcgacgtg 120gtgctgcacg cattggccga cgccatcttg ggagccgccg ctttgggcga catcggcaaa 180catttcccgg acaccgaccc caatttcaag ggcgccgaca gcagggtgct actgcgccac 240gtgtacggca tcgtcaagga aaaaggctat aaactggtca acgccgacgt gaccatcatc 300gctcaggcgc cgaagatgct gccacacgtg cccggcatgc gcgccaacat tgccgccgat 360ctggaaaccg atgtcgattt cattaatgta aaagccacga cgaccgagaa actgggcttt 420gagggccgta aggaaggcat cgccgtgcag gctgtggtgt tgatagaacg c 471 10 157 PRTMethylomonas 16a Amino acid sequences encoded by ORF5 10 Met Ile Arg ValGly Met Gly Tyr Asp Val His Arg Phe Asn Asp Gly 1 5 10 15 Asp His IleIle Leu Gly Gly Val Lys Ile Pro Tyr Glu Lys Gly Leu 20 25 30 Glu Ala HisSer Asp Gly Asp Val Val Leu His Ala Leu Ala Asp Ala 35 40 45 Ile Leu GlyAla Ala Ala Leu Gly Asp Ile Gly Lys His Phe Pro Asp 50 55 60 Thr Asp ProAsn Phe Lys Gly Ala Asp Ser Arg Val Leu Leu Arg His 65 70 75 80 Val TyrGly Ile Val Lys Glu Lys Gly Tyr Lys Leu Val Asn Ala Asp 85 90 95 Val ThrIle Ile Ala Gln Ala Pro Lys Met Leu Pro His Val Pro Gly 100 105 110 MetArg Ala Asn Ile Ala Ala Asp Leu Glu Thr Asp Val Asp Phe Ile 115 120 125Asn Val Lys Ala Thr Thr Thr Glu Lys Leu Gly Phe Glu Gly Arg Lys 130 135140 Glu Gly Ile Ala Val Gln Ala Val Val Leu Ile Glu Arg 145 150 155 111632 DNA Methylomonas 16a ORF6 11 atgacaaaat tcatctttat caccggcggcgtggtgtcat ccttgggaaa agggatagcc 60 gcctcctccc tggcggcgat tctggaagaccgcggcctca aagtcactat cacaaaactc 120 gatccctaca tcaacgtcga ccccggcaccatgagcccgt ttcaacacgg cgaggtgttc 180 gtgaccgaag acggtgccga aaccgatttggaccttggcc attacgaacg gtttttgaaa 240 accacgatga ccaagaaaaa caacttcaccaccggtcagg tttacgagca ggtattacgc 300 aacgagcgca aaggtgatta tcttggcgcgaccgtgcaag tcattccaca tatcaccgac 360 gaaatcaaac gccgggtgta tgaaagcgccgaagggaaag atgtggcatt gatcgaagtc 420 ggcggcacgg tgggcgacat cgaatcgttaccgtttctgg aaaccatacg ccagatgggc 480 gtggaactgg gtcgtgaccg cgccttgttcattcatttga cgctggtgcc ttacatcaaa 540 tcggccggcg aactgaaaac caagcccacccagcattcgg tcaaagaact gcgcaccatc 600 gggattcagc cggacatttt gatctgtcgttcagaacaac cgatcccggc cagtgaacgc 660 cgcaagatcg cgctatttac caatgtcgccgaaaaggcgg tgatttccgc gatcgatgcc 720 gacaccattt accgcattcc gctattgctgcgcgaacaag gcctggacga cctggtggtc 780 gatcagttgc gcctggacgt accagcggcggatttatcgg cctgggaaaa ggtcgtcgat 840 ggcctgactc atccgaccga cgaagtcagcattgcgatcg tcggtaaata tgtcgaccac 900 accgatgcct acaaatcgct gaatgaagccctgattcatg ccggcattca cacgcgccac 960 aaggtgcaaa tcagctacat cgactccgaaaccatagaag ccgaaggcac cgccaaattg 1020 aaaaacgtcg atgcgatcct ggtgccgggtggtttcggcg aacgcggcgt ggaaggcaag 1080 atttctaccg tgcgttttgc ccgcgagaacaaaatcccgt atttgggcat ttgcttgggc 1140 atgcaatcgg cggtaatcga attcgcccgcaacgtggttg gcctggaagg cgcgcacagc 1200 accgaattcc tgccgaaatc gccacaccctgtgatcggct tgatcaccga atggatggac 1260 gaagccggcg aactggtcac acgcgacgaagattccgatc tgggcggcac gatgcgtctg 1320 ggcgcgcaaa aatgccgcct gaaggctgattccttggctt ttcagttgta tcaaaaagac 1380 gtcatcaccg agcgtcaccg ccaccgctacgaattcaaca atcaatattt aaaacaactg 1440 gaagcggccg gcatgaaatt ttccggtaaatcgctggacg gccgcctggt ggagatcatc 1500 gagctacccg aacacccctg gttcctggcctgccagttcc atcccgaatt cacctcgacg 1560 ccgcgtaacg gccacgccct attttcgggcttcgtcgaag cggccgccaa acacaaaaca 1620 caaggcacag ca 1632 12 544 PRTMethylomonas 16a Amino acid sequences encoded by ORF6 12 Met Thr Lys PheIle Phe Ile Thr Gly Gly Val Val Ser Ser Leu Gly 1 5 10 15 Lys Gly IleAla Ala Ser Ser Leu Ala Ala Ile Leu Glu Asp Arg Gly 20 25 30 Leu Lys ValThr Ile Thr Lys Leu Asp Pro Tyr Ile Asn Val Asp Pro 35 40 45 Gly Thr MetSer Pro Phe Gln His Gly Glu Val Phe Val Thr Glu Asp 50 55 60 Gly Ala GluThr Asp Leu Asp Leu Gly His Tyr Glu Arg Phe Leu Lys 65 70 75 80 Thr ThrMet Thr Lys Lys Asn Asn Phe Thr Thr Gly Gln Val Tyr Glu 85 90 95 Gln ValLeu Arg Asn Glu Arg Lys Gly Asp Tyr Leu Gly Ala Thr Val 100 105 110 GlnVal Ile Pro His Ile Thr Asp Glu Ile Lys Arg Arg Val Tyr Glu 115 120 125Ser Ala Glu Gly Lys Asp Val Ala Leu Ile Glu Val Gly Gly Thr Val 130 135140 Gly Asp Ile Glu Ser Leu Pro Phe Leu Glu Thr Ile Arg Gln Met Gly 145150 155 160 Val Glu Leu Gly Arg Asp Arg Ala Leu Phe Ile His Leu Thr LeuVal 165 170 175 Pro Tyr Ile Lys Ser Ala Gly Glu Leu Lys Thr Lys Pro ThrGln His 180 185 190 Ser Val Lys Glu Leu Arg Thr Ile Gly Ile Gln Pro AspIle Leu Ile 195 200 205 Cys Arg Ser Glu Gln Pro Ile Pro Ala Ser Glu ArgArg Lys Ile Ala 210 215 220 Leu Phe Thr Asn Val Ala Glu Lys Ala Val IleSer Ala Ile Asp Ala 225 230 235 240 Asp Thr Ile Tyr Arg Ile Pro Leu LeuLeu Arg Glu Gln Gly Leu Asp 245 250 255 Asp Leu Val Val Asp Gln Leu ArgLeu Asp Val Pro Ala Ala Asp Leu 260 265 270 Ser Ala Trp Glu Lys Val ValAsp Gly Leu Thr His Pro Thr Asp Glu 275 280 285 Val Ser Ile Ala Ile ValGly Lys Tyr Val Asp His Thr Asp Ala Tyr 290 295 300 Lys Ser Leu Asn GluAla Leu Ile His Ala Gly Ile His Thr Arg His 305 310 315 320 Lys Val GlnIle Ser Tyr Ile Asp Ser Glu Thr Ile Glu Ala Glu Gly 325 330 335 Thr AlaLys Leu Lys Asn Val Asp Ala Ile Leu Val Pro Gly Gly Phe 340 345 350 GlyGlu Arg Gly Val Glu Gly Lys Ile Ser Thr Val Arg Phe Ala Arg 355 360 365Glu Asn Lys Ile Pro Tyr Leu Gly Ile Cys Leu Gly Met Gln Ser Ala 370 375380 Val Ile Glu Phe Ala Arg Asn Val Val Gly Leu Glu Gly Ala His Ser 385390 395 400 Thr Glu Phe Leu Pro Lys Ser Pro His Pro Val Ile Gly Leu IleThr 405 410 415 Glu Trp Met Asp Glu Ala Gly Glu Leu Val Thr Arg Asp GluAsp Ser 420 425 430 Asp Leu Gly Gly Thr Met Arg Leu Gly Ala Gln Lys CysArg Leu Lys 435 440 445 Ala Asp Ser Leu Ala Phe Gln Leu Tyr Gln Lys AspVal Ile Thr Glu 450 455 460 Arg His Arg His Arg Tyr Glu Phe Asn Asn GlnTyr Leu Lys Gln Leu 465 470 475 480 Glu Ala Ala Gly Met Lys Phe Ser GlyLys Ser Leu Asp Gly Arg Leu 485 490 495 Val Glu Ile Ile Glu Leu Pro GluHis Pro Trp Phe Leu Ala Cys Gln 500 505 510 Phe His Pro Glu Phe Thr SerThr Pro Arg Asn Gly His Ala Leu Phe 515 520 525 Ser Gly Phe Val Glu AlaAla Ala Lys His Lys Thr Gln Gly Thr Ala 530 535 540 13 891 DNAMethylomonas 16a ORF7 13 atgagtaaat tgaaagccta cctgaccgtc tgccaagaacgcgtcgagcg cgcgctggac 60 gcccgtctgc ctgccgaaaa catactgcca caaaccttgcatcaggccat gcgctattcc 120 gtattgaacg gcggcaaacg cacccggccc ttgttgacttatgcgaccgg tcaggctttg 180 ggcttgccgg aaaacgtgct ggatgcgccg gcttgcgcggtagaattcat ccatgtgtat 240 tcgctgattc acgacgatct gccggccatg gacaacgatgatctgcgccg cggcaaaccg 300 acctgtcaca aggcttacga cgaggccacc gccattttggccggcgacgc actgcaggcg 360 ctggcctttg aagttctggc caacgacccc ggcatcaccgtcgatgcccc ggctcgcctg 420 aaaatgatca cggctttgac ccgcgccagc ggctctcaaggcatggtggg cggtcaagcc 480 atcgatctcg gctccgtcgg ccgcaaattg acgctgccggaactcgaaaa catgcatatc 540 cacaagactg gcgccctgat ccgcgccagc gtcaatctggcggcattatc caaacccgat 600 ctggatactt gcgtcgccaa gaaactggat cactatgccaaatgcatagg cttgtcgttc 660 caggtcaaag acgacattct cgacatcgaa gccgacaccgcgacactcgg caagactcag 720 ggcaaggaca tcgataacga caaaccgacc taccctgcgctattgggcat ggctggcgcc 780 aaacaaaaag cccaggaatt gcacgaacaa gcagtcgaaagcttaacggg atttggcagc 840 gaagccgacc tgctgcgcga actatcgctt tacatcatcgagcgcacgca c 891 14 297 PRT Methylomonas 16a Amino acid sequencesencoded by ORF7 14 Met Ser Lys Leu Lys Ala Tyr Leu Thr Val Cys Gln GluArg Val Glu 1 5 10 15 Arg Ala Leu Asp Ala Arg Leu Pro Ala Glu Asn IleLeu Pro Gln Thr 20 25 30 Leu His Gln Ala Met Arg Tyr Ser Val Leu Asn GlyGly Lys Arg Thr 35 40 45 Arg Pro Leu Leu Thr Tyr Ala Thr Gly Gln Ala LeuGly Leu Pro Glu 50 55 60 Asn Val Leu Asp Ala Pro Ala Cys Ala Val Glu PheIle His Val Tyr 65 70 75 80 Ser Leu Ile His Asp Asp Leu Pro Ala Met AspAsn Asp Asp Leu Arg 85 90 95 Arg Gly Lys Pro Thr Cys His Lys Ala Tyr AspGlu Ala Thr Ala Ile 100 105 110 Leu Ala Gly Asp Ala Leu Gln Ala Leu AlaPhe Glu Val Leu Ala Asn 115 120 125 Asp Pro Gly Ile Thr Val Asp Ala ProAla Arg Leu Lys Met Ile Thr 130 135 140 Ala Leu Thr Arg Ala Ser Gly SerGln Gly Met Val Gly Gly Gln Ala 145 150 155 160 Ile Asp Leu Gly Ser ValGly Arg Lys Leu Thr Leu Pro Glu Leu Glu 165 170 175 Asn Met His Ile HisLys Thr Gly Ala Leu Ile Arg Ala Ser Val Asn 180 185 190 Leu Ala Ala LeuSer Lys Pro Asp Leu Asp Thr Cys Val Ala Lys Lys 195 200 205 Leu Asp HisTyr Ala Lys Cys Ile Gly Leu Ser Phe Gln Val Lys Asp 210 215 220 Asp IleLeu Asp Ile Glu Ala Asp Thr Ala Thr Leu Gly Lys Thr Gln 225 230 235 240Gly Lys Asp Ile Asp Asn Asp Lys Pro Thr Tyr Pro Ala Leu Leu Gly 245 250255 Met Ala Gly Ala Lys Gln Lys Ala Gln Glu Leu His Glu Gln Ala Val 260265 270 Glu Ser Leu Thr Gly Phe Gly Ser Glu Ala Asp Leu Leu Arg Glu Leu275 280 285 Ser Leu Tyr Ile Ile Glu Arg Thr His 290 295 15 1533 DNAMethylomonas 16a ORF8 15 atggccaaca ccaaacacat catcatcgtc ggcgcgggtcccggcggact ttgcgccggc 60 atgttgctga gccagcgcgg cttcaaggta tcgattttcgacaaacatgc agaaatcggc 120 ggccgcaacc gcccgatcaa catgaacggc tttaccttcgataccggtcc gacattcttg 180 ttgatgaaag gcgtgctgga cgaaatgttc gaactgtgcgagcgccgtag cgaggattat 240 ctggaattcc tgccgctaag cccgatgtac cgcctgctgtacgacgaccg cgacatcttc 300 gtctattccg accgcgagaa catgcgcgcc gaattgcaacgggtattcga cgaaggcacg 360 gacggctacg aacagttcat ggaacaggaa cgcaaacgcttcaacgcgct gtatccctgc 420 atcacccgcg attattccag cctgaaatcc tttttgtcgctggacttgat caaggccctg 480 ccgtggctgg cttttccgaa aagcgtgttc aataatctcggccagtattt caaccaggaa 540 aaaatgcgcc tggccttttg ctttcagtcc aagtatctgggcatgtcgcc gtgggaatgc 600 ccggcactgt ttacgatgct gccctatctg gagcacgaatacggcattta tcacgtcaaa 660 ggcggcctga accgcatcgc ggcggcgatg gcgcaagtgatcgcggaaaa cggcggcgaa 720 attcacttga acagcgaaat cgagtcgctg atcatcgaaaacggcgctgc caagggcgtc 780 aaattacaac atggcgcgga gctgcgcggc gacgaagtcatcatcaacgc ggattttgcc 840 cacgcgatga cgcatctggt caaaccgggc gtcttgaaaaaatacacccc ggaaaacctg 900 aagcagcgcg agtattcctg ttcgaccttc atgctgtatctgggtttgga caagatttac 960 gatctgccgc accataccat cgtgtttgcc aaggattacaccaccaatat ccgcaacatt 1020 ttcgacaaca aaaccctgac ggacgatttt tcgttttacgtgcaaaacgc cagcgccagc 1080 gacgacagcc tagcgccagc cggcaaatcg gcgctgtacgtgctggtgcc gatgcccaac 1140 aacgacagcg gcctggactg gcaggcgcat tgccaaaacgtgcgcgaaca ggtgttggac 1200 acgctgggcg cgcgactggg attgagcgac atcagagcccatatcgaatg cgaaaaaatc 1260 atcacgccgc aaacctggga aacggacgaa cacgtttacaagggcgccac tttcagtttg 1320 tcgcacaagt tcagccaaat gctgtactgg cggccgcacaaccgtttcga ggaactggcc 1380 aattgctatc tggtcggcgg cggcacgcat cccggtagcggtttgccgac catctacgaa 1440 tcggcgcgga tttcggccaa gctgatttcc cagaaacatcgggtgaggtt caaggacata 1500 gcacacagcg cctggctgaa aaaagccaaa gcc 1533 16511 PRT Methylomonas 16a Amino acid sequences encoded by ORF8 16 Met AlaAsn Thr Lys His Ile Ile Ile Val Gly Ala Gly Pro Gly Gly 1 5 10 15 LeuCys Ala Gly Met Leu Leu Ser Gln Arg Gly Phe Lys Val Ser Ile 20 25 30 PheAsp Lys His Ala Glu Ile Gly Gly Arg Asn Arg Pro Ile Asn Met 35 40 45 AsnGly Phe Thr Phe Asp Thr Gly Pro Thr Phe Leu Leu Met Lys Gly 50 55 60 ValLeu Asp Glu Met Phe Glu Leu Cys Glu Arg Arg Ser Glu Asp Tyr 65 70 75 80Leu Glu Phe Leu Pro Leu Ser Pro Met Tyr Arg Leu Leu Tyr Asp Asp 85 90 95Arg Asp Ile Phe Val Tyr Ser Asp Arg Glu Asn Met Arg Ala Glu Leu 100 105110 Gln Arg Val Phe Asp Glu Gly Thr Asp Gly Tyr Glu Gln Phe Met Glu 115120 125 Gln Glu Arg Lys Arg Phe Asn Ala Leu Tyr Pro Cys Ile Thr Arg Asp130 135 140 Tyr Ser Ser Leu Lys Ser Phe Leu Ser Leu Asp Leu Ile Lys AlaLeu 145 150 155 160 Pro Trp Leu Ala Phe Pro Lys Ser Val Phe Asn Asn LeuGly Gln Tyr 165 170 175 Phe Asn Gln Glu Lys Met Arg Leu Ala Phe Cys PheGln Ser Lys Tyr 180 185 190 Leu Gly Met Ser Pro Trp Glu Cys Pro Ala LeuPhe Thr Met Leu Pro 195 200 205 Tyr Leu Glu His Glu Tyr Gly Ile Tyr HisVal Lys Gly Gly Leu Asn 210 215 220 Arg Ile Ala Ala Ala Met Ala Gln ValIle Ala Glu Asn Gly Gly Glu 225 230 235 240 Ile His Leu Asn Ser Glu IleGlu Ser Leu Ile Ile Glu Asn Gly Ala 245 250 255 Ala Lys Gly Val Lys LeuGln His Gly Ala Glu Leu Arg Gly Asp Glu 260 265 270 Val Ile Ile Asn AlaAsp Phe Ala His Ala Met Thr His Leu Val Lys 275 280 285 Pro Gly Val LeuLys Lys Tyr Thr Pro Glu Asn Leu Lys Gln Arg Glu 290 295 300 Tyr Ser CysSer Thr Phe Met Leu Tyr Leu Gly Leu Asp Lys Ile Tyr 305 310 315 320 AspLeu Pro His His Thr Ile Val Phe Ala Lys Asp Tyr Thr Thr Asn 325 330 335Ile Arg Asn Ile Phe Asp Asn Lys Thr Leu Thr Asp Asp Phe Ser Phe 340 345350 Tyr Val Gln Asn Ala Ser Ala Ser Asp Asp Ser Leu Ala Pro Ala Gly 355360 365 Lys Ser Ala Leu Tyr Val Leu Val Pro Met Pro Asn Asn Asp Ser Gly370 375 380 Leu Asp Trp Gln Ala His Cys Gln Asn Val Arg Glu Gln Val LeuAsp 385 390 395 400 Thr Leu Gly Ala Arg Leu Gly Leu Ser Asp Ile Arg AlaHis Ile Glu 405 410 415 Cys Glu Lys Ile Ile Thr Pro Gln Thr Trp Glu ThrAsp Glu His Val 420 425 430 Tyr Lys Gly Ala Thr Phe Ser Leu Ser His LysPhe Ser Gln Met Leu 435 440 445 Tyr Trp Arg Pro His Asn Arg Phe Glu GluLeu Ala Asn Cys Tyr Leu 450 455 460 Val Gly Gly Gly Thr His Pro Gly SerGly Leu Pro Thr Ile Tyr Glu 465 470 475 480 Ser Ala Arg Ile Ser Ala LysLeu Ile Ser Gln Lys His Arg Val Arg 485 490 495 Phe Lys Asp Ile Ala HisSer Ala Trp Leu Lys Lys Ala Lys Ala 500 505 510 17 1491 DNA Methylomonas16a ORF9 17 atgaactcaa atgacaacca acgcgtgatc gtgatcggcg ccggcctcggcggcctgtcc 60 gccgctattt cgctggccac ggccggcttt tccgtgcaac tcatcgaaaaaaacgacaag 120 gtcggcggca agctcaacat catgaccaaa gacggcttta ccttcgatctggggccgtcc 180 attttgacga tgccgcacat ctttgaggcc ttgttcacag gggccggcaaaaacatggcc 240 gattacgtgc aaatccagaa agtcgaaccg cactggcgca atttcttcgaggacggtagc 300 gtgatcgact tgtgcgaaga cgccgaaacc cagcgccgcg agctggataaacttggcccc 360 ggcacttacg cgcaattcca gcgctttctg gactattcga aaaacctctgcacggaaacc 420 gaagccggtt acttcgccaa gggcctggac ggcttttggg atttactcaagttttacggc 480 ccgctccgca gcctgctgag tttcgacgtc ttccgcagca tggaccagggcgtgcgccgc 540 tttatttccg atcccaagtt ggtcgaaatc ctgaattact tcatcaaatacgtcggctcc 600 tcgccttacg atgcgcccgc cttgatgaac ctgctgcctt acattcaatatcattacggc 660 ctgtggtacg tgaaaggcgg catgtatggc atggcgcagg ccatggaaaaactggccgtg 720 gaattgggcg tcgagattcg tttagatgcc gaggtgtcgg aaatccaaaaacaggacggc 780 agagcctgcg ccgtaaagtt ggcgaacggc gacgtgctgc cggccgacatcgtggtgtcg 840 aacatggaag tgattccggc gatggaaaaa ctgctgcgca gcccggccagcgaactgaaa 900 aaaatgcagc gcttcgagcc tagctgttcc ggcctggtgc tgcacttgggcgtggacagg 960 ctgtatccgc aactggcgca ccacaatttc ttttattccg atcatccgcgcgaacatttc 1020 gatgcggtat tcaaaagcca tcgcctgtcg gacgatccga ccatttatctggtcgcgccg 1080 tgcaagaccg accccgccca ggcgccggcc ggctgcgaga tcatcaaaatcctgccccat 1140 atcccgcacc tcgaccccga caaactgctg accgccgagg attattcagccttgcgcgag 1200 cgggtgctgg tcaaactcga acgcatgggc ctgacggatt tacgccaacacatcgtgacc 1260 gaagaatact ggacgccgct ggatattcag gccaaatatt attcaaaccagggctcgatt 1320 tacggcgtgg tcgccgaccg cttcaaaaac ctgggtttca aggcacctcaacgcagcagc 1380 gaattatcca atctgtattt cgtcggcggc agcgtcaatc ccggcggcggcatgccgatg 1440 gtgacgctgt ccgggcaatt ggtgagggac aagattgtgg cggatttgca a1491 18 497 PRT Methylomonas 16a Amino acid sequences encoded by ORF9 18Met Asn Ser Asn Asp Asn Gln Arg Val Ile Val Ile Gly Ala Gly Leu 1 5 1015 Gly Gly Leu Ser Ala Ala Ile Ser Leu Ala Thr Ala Gly Phe Ser Val 20 2530 Gln Leu Ile Glu Lys Asn Asp Lys Val Gly Gly Lys Leu Asn Ile Met 35 4045 Thr Lys Asp Gly Phe Thr Phe Asp Leu Gly Pro Ser Ile Leu Thr Met 50 5560 Pro His Ile Phe Glu Ala Leu Phe Thr Gly Ala Gly Lys Asn Met Ala 65 7075 80 Asp Tyr Val Gln Ile Gln Lys Val Glu Pro His Trp Arg Asn Phe Phe 8590 95 Glu Asp Gly Ser Val Ile Asp Leu Cys Glu Asp Ala Glu Thr Gln Arg100 105 110 Arg Glu Leu Asp Lys Leu Gly Pro Gly Thr Tyr Ala Gln Phe GlnArg 115 120 125 Phe Leu Asp Tyr Ser Lys Asn Leu Cys Thr Glu Thr Glu AlaGly Tyr 130 135 140 Phe Ala Lys Gly Leu Asp Gly Phe Trp Asp Leu Leu LysPhe Tyr Gly 145 150 155 160 Pro Leu Arg Ser Leu Leu Ser Phe Asp Val PheArg Ser Met Asp Gln 165 170 175 Gly Val Arg Arg Phe Ile Ser Asp Pro LysLeu Val Glu Ile Leu Asn 180 185 190 Tyr Phe Ile Lys Tyr Val Gly Ser SerPro Tyr Asp Ala Pro Ala Leu 195 200 205 Met Asn Leu Leu Pro Tyr Ile GlnTyr His Tyr Gly Leu Trp Tyr Val 210 215 220 Lys Gly Gly Met Tyr Gly MetAla Gln Ala Met Glu Lys Leu Ala Val 225 230 235 240 Glu Leu Gly Val GluIle Arg Leu Asp Ala Glu Val Ser Glu Ile Gln 245 250 255 Lys Gln Asp GlyArg Ala Cys Ala Val Lys Leu Ala Asn Gly Asp Val 260 265 270 Leu Pro AlaAsp Ile Val Val Ser Asn Met Glu Val Ile Pro Ala Met 275 280 285 Glu LysLeu Leu Arg Ser Pro Ala Ser Glu Leu Lys Lys Met Gln Arg 290 295 300 PheGlu Pro Ser Cys Ser Gly Leu Val Leu His Leu Gly Val Asp Arg 305 310 315320 Leu Tyr Pro Gln Leu Ala His His Asn Phe Phe Tyr Ser Asp His Pro 325330 335 Arg Glu His Phe Asp Ala Val Phe Lys Ser His Arg Leu Ser Asp Asp340 345 350 Pro Thr Ile Tyr Leu Val Ala Pro Cys Lys Thr Asp Pro Ala GlnAla 355 360 365 Pro Ala Gly Cys Glu Ile Ile Lys Ile Leu Pro His Ile ProHis Leu 370 375 380 Asp Pro Asp Lys Leu Leu Thr Ala Glu Asp Tyr Ser AlaLeu Arg Glu 385 390 395 400 Arg Val Leu Val Lys Leu Glu Arg Met Gly LeuThr Asp Leu Arg Gln 405 410 415 His Ile Val Thr Glu Glu Tyr Trp Thr ProLeu Asp Ile Gln Ala Lys 420 425 430 Tyr Tyr Ser Asn Gln Gly Ser Ile TyrGly Val Val Ala Asp Arg Phe 435 440 445 Lys Asn Leu Gly Phe Lys Ala ProGln Arg Ser Ser Glu Leu Ser Asn 450 455 460 Leu Tyr Phe Val Gly Gly SerVal Asn Pro Gly Gly Gly Met Pro Met 465 470 475 480 Val Thr Leu Ser GlyGln Leu Val Arg Asp Lys Ile Val Ala Asp Leu 485 490 495 Gln 19 22 DNAArtificial Sequence Description of Artificial Sequenceprimer 19aaggatccgc gtattcgtac tc 22 20 40 DNA Artificial Sequence Description ofArtificial Sequenceprimer 20 ctggatccga tctagaaata ggctcgagtt gtcgttcagg40 21 30 DNA Artificial Sequence Description of ArtificialSequenceprimer 21 aaggatccta ctcgagctga catcagtgct 30 22 22 DNAArtificial Sequence Description of Artificial Sequenceprimer 22gctctagatg caaccagaat cg 22 23 954 DNA Methylomonas 16a 23 atgcaaatcgtactcgcaaa cccccgtgga ttctgtgccg gcgtggaccg ggccattgaa 60 attgtcgatcaagccatcga agcctttggt gcgccgattt atgtgcggca cgaggtggtg 120 cataaccgcaccgtggtcga tggactgaaa caaaaaggtg cggtgttcat cgaggaacta 180 agcgatgtgccggtgggttc ctacttgatt ttcagcgcgc acggcgtatc caaggaggtg 240 caacaggaagccgaggagcg ccagttgacg gtattcgatg cgacttgtcc gctggtgacc 300 aaagtgcacatgcaggttgc caagcatgcc aaacagggcc gagaagtgat tttgatcggc 360 cacgccggtcatccggaagt ggaaggcacg atgggccagt atgaaaaatg caccgaaggc 420 ggcggcatttatctggtcga aactccggaa gacgtacgca atttgaaagt caacaatccc 480 aatgatctggcctatgtgac gcagacgacc ttgtcgatga ccgacaccaa ggtcatggtg 540 gatgcgttacgcgaacaatt tccgtccatt aaggagcaaa aaaaggacga tatttgttac 600 gcgacgcaaaaccgtcagga tgcggtgcat gatctggcca agatttccga cctgattctg 660 gttgtcggctctcccaatag ttcgaattcc aaccgtttgc gtgaaatcgc cgtgcaactc 720 ggtaaacccgcttatttgat cgatacttac caggatttga agcaagattg gctggaggga 780 attgaagtagtcggggttac cgcgggcgct tcggcgccgg aagtgttggt gcaggaagtg 840 atcgatcaactgaaggcatg gggcggcgaa accacttcgg tcagagaaaa cagcggcatc 900 gaggaaaaggtagtcttttc gattcccaag gagttgaaaa aacatatgca agcg 954 24 318 PRTMethylomonas 16a 24 Met Gln Ile Val Leu Ala Asn Pro Arg Gly Phe Cys AlaGly Val Asp 1 5 10 15 Arg Ala Ile Glu Ile Val Asp Gln Ala Ile Glu AlaPhe Gly Ala Pro 20 25 30 Ile Tyr Val Arg His Glu Val Val His Asn Arg ThrVal Val Asp Gly 35 40 45 Leu Lys Gln Lys Gly Ala Val Phe Ile Glu Glu LeuSer Asp Val Pro 50 55 60 Val Gly Ser Tyr Leu Ile Phe Ser Ala His Gly ValSer Lys Glu Val 65 70 75 80 Gln Gln Glu Ala Glu Glu Arg Gln Leu Thr ValPhe Asp Ala Thr Cys 85 90 95 Pro Leu Val Thr Lys Val His Met Gln Val AlaLys His Ala Lys Gln 100 105 110 Gly Arg Glu Val Ile Leu Ile Gly His AlaGly His Pro Glu Val Glu 115 120 125 Gly Thr Met Gly Gln Tyr Glu Lys CysThr Glu Gly Gly Gly Ile Tyr 130 135 140 Leu Val Glu Thr Pro Glu Asp ValArg Asn Leu Lys Val Asn Asn Pro 145 150 155 160 Asn Asp Leu Ala Tyr ValThr Gln Thr Thr Leu Ser Met Thr Asp Thr 165 170 175 Lys Val Met Val AspAla Leu Arg Glu Gln Phe Pro Ser Ile Lys Glu 180 185 190 Gln Lys Lys AspAsp Ile Cys Tyr Ala Thr Gln Asn Arg Gln Asp Ala 195 200 205 Val His AspLeu Ala Lys Ile Ser Asp Leu Ile Leu Val Val Gly Ser 210 215 220 Pro AsnSer Ser Asn Ser Asn Arg Leu Arg Glu Ile Ala Val Gln Leu 225 230 235 240Gly Lys Pro Ala Tyr Leu Ile Asp Thr Tyr Gln Asp Leu Lys Gln Asp 245 250255 Trp Leu Glu Gly Ile Glu Val Val Gly Val Thr Ala Gly Ala Ser Ala 260265 270 Pro Glu Val Leu Val Gln Glu Val Ile Asp Gln Leu Lys Ala Trp Gly275 280 285 Gly Glu Thr Thr Ser Val Arg Glu Asn Ser Gly Ile Glu Glu LysVal 290 295 300 Val Phe Ser Ile Pro Lys Glu Leu Lys Lys His Met Gln Ala305 310 315

What is claimed is:
 1. An isolated nucleic acid molecule encoding aisoprenoid biosynthetic enzyme, selected from the group consisting of:(a) an isolated nucleic acid molecule encoding the amino acid sequenceselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,16, 18 and 24; (b) an isolated nucleic acid molecule that hybridizeswith (a) under the following hybridization conditions: 0.1×SSC, 0.1%SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%SDS; and (c) an isolated nucleic acid molecule that is complementary to(a) or (b).
 2. The isolated nucleic acid molecule of claim 1 selectedfrom the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17and
 23. 3. A polypeptide encoded by the isolated nucleic acid moleculeof claim
 1. 4. The polypeptide of claim 3 selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and
 18. 5. Anisolated nucleic acid molecule comprising a first nucleotide sequenceencoding a polypeptide of at least 620 amino acids that has at least 60%identity based on the Smith-Waterman method of alignment when comparedto a polypeptide having the sequence as set forth in SEQ ID NO:2 or asecond nucleotide sequence comprising the complement of the firstnucleotide sequence.
 6. An isolated nucleic acid molecule comprising afirst nucleotide sequence encoding a polypeptide of at least 394 aminoacids that has at least 55% identity based on the Smith-Waterman methodof alignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:4 or a second nucleotide sequence comprising thecomplement of the first nucleotide sequence.
 7. An isolated nucleic acidmolecule comprising a first nucleotide sequence encoding a polypeptideof at least 231 amino acids that has at least 52% identity based on theSmith-Waterman method of alignment when compared to a polypeptide havingthe sequence as set forth in SEQ ID NO:6 or a second nucleotide sequencecomprising the complement of the first nucleotide sequence.
 8. Anisolated nucleic acid molecule comprising a first nucleotide sequenceencoding a polypeptide of at least 285 amino acids that has at least 50%identity based on the Smith-Waterman method of alignment when comparedto a polypeptide having the sequence as set forth in SEQ ID NO:8 or asecond nucleotide sequence comprising the complement of the firstnucleotide sequence.
 9. An isolated nucleic acid molecule comprising afirst nucleotide sequence encoding a polypeptide of at least 157 aminoacids that has at least 69% identity based on the Smith-Waterman methodof alignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:10 or a second nucleotide sequence comprising thecomplement of the first nucleotide sequence.
 10. An isolated nucleicacid molecule comprising a first nucleotide sequence encoding apolypeptide of at least 544 amino acids that has at least 67% identitybased on the Smith-Waterman method of alignment when compared to apolypeptide having the sequence as set forth in SEQ ID NO:12 or a secondnucleotide sequence comprising the complement of the first nucleotidesequence.
 11. An isolated nucleic acid molecule comprising a firstnucleotide sequence encoding a polypeptide of at least 297 amino acidsthat has at least 57% identity based on the Smith-Waterman method ofalignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:14 or a second nucleotide sequence comprising thecomplement of the first nucleotide sequence.
 12. An isolated nucleicacid molecule comprising a first nucleotide sequence encoding apolypeptide of at least 511 amino acids that has at least 34% identitybased on the Smith-Waterman method of alignment when compared to apolypeptide having the sequence as set forth in SEQ ID NO:16 or a secondnucleotide sequence comprising the complement of the first nucleotidesequence.
 13. An isolated nucleic acid molecule comprising a firstnucleotide sequence encoding a polypeptide of at least 497 amino acidsthat has at least 49% identity based on the Smith-Waterman method ofalignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:18 or a second nucleotide sequence comprising thecomplement of the first nucleotide sequence.
 14. An isolated nucleicacid molecule comprising a first nucleotide sequence encoding apolypeptide of at least 318 amino acids that has at least 65% identitybased on the Smith-Waterman method of alignment when compared to apolypeptide having the sequence as set forth in SEQ ID NO:24 or a secondnucleotide sequence comprising the complement of the first nucleotidesequence.
 15. A chimeric gene comprising the isolated nucleic acidmolecule of any one of claims 1 or 5-14 operably linked to suitableregulatory sequences.
 16. A transformed host cell comprising thechimeric gene of claim
 15. 17. The transformed host cell of claim 16wherein the host cell is selected from the group consisting of bacteria,yeast, filamentous fungi, and green plants.
 18. The transformed hostcell of claim 17 wherein the host cell is selected from the groupconsisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida,Hansenula, Salmonella, Bacillus, Acinetobacter, Rhodococcus,Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter,Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium andKlebsiella.
 19. The transformed host cell of claim 17 wherein the hostcell is selected from the group consisting of soybean, rapeseed,sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum,rice, Arabidopsis, cruciferous vegetables, melons, carrots, celery,parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seedcrops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees,softwood trees, and forage grasses.
 20. A method of obtaining a nucleicacid molecule encoding an isoprenoid compound biosynthetic enzymecomprising: (a) probing a genomic library with the nucleic acid moleculeof any one of claims 1 or 5-14; (b) identifying a DNA clone thathybridizes with the nucleic acid molecule of any one of claims 1 or5-14; and (c) sequencing the genomic fragment that comprises the cloneidentified in step (b), wherein the sequenced genomic fragment encodesan isoprenoid biosynthetic enzyme.
 21. A method of obtaining a nucleicacid molecule encoding an isoprenoid biosynthetic enzyme comprising: (a)synthesizing an at least one oligonucleotide primer corresponding to aportion of the sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17 and 23; and (b) amplifying an insertpresent in a cloning vector using the oligonucleotide primer of step(a); wherein the amplified insert encodes a portion of an amino acidsequence encoding an isoprenoid biosynthetic enzyme.
 22. The product ofthe method of claims 20 or
 21. 23. A method for the production ofisoprenoid compounds comprising: contacting a transformed host cellunder suitable growth conditions with an effective amount of a carbonsource whereby an isoprenoid compound is produced, said transformed hostcell comprising a set of nucleic acid molecules encoding SEQ ID NOs:2,4, 6, 8, 10, 12, 14, 16, 18 and 24 under the control of suitableregulatory sequences.
 24. A method according to claim 23 wherein thetransformed host cell is selected form the group consisting ofAspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula,Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces,Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes,Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella.25. A method according to claim 23 wherein said methanotrophic bacteria:(a) grows on a C1 carbon substrate selected from the group consisting ofmethane and methanol; and (b) comprises a functional Embden-Meyerofcarbon pathway, said pathway comprising a gene encoding a pyrophosphatedependent phosphofructokinase enzyme.
 26. A method according to claim 25wherein said methanotrophic bacteria is methylomonas 16a ATCC PTA 2402.27. A method according to claim 23 wherein the transformed host cell isselected form the group consisting of soybean, rapeseed, sunflower,cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice,Arabidopsis, cruciferous vegetables, melons, carrots, celery, parsley,tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops,sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees,softwood trees, and forage grasses.
 28. A method according to claim 23wherein the carbon source is selected from the group consisting ofmonosaccharides, oligosaccharides, polysaccharides, carbon dioxide,methanol, methane, formaldehyde, formate, and carbon-containing amines.29. A method according to claim 23 wherein the transformed host isselected from the group consisting of Methylomonas, Methylobacter andMethanobacterium and the carbon source is selected from the groupconsisting of methane and methanol.
 30. A method of regulatingisoprenoid biosynthesis in an organism comprising, over-expressing atleast one isoprenoid gene selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17 and 23 in an organism such that theisoprenoid biosynthesis is altered in the organism.
 31. A methodaccording to claim 30 wherein said isoprenoid gene is over-expressed ona multicopy plasmid.
 32. A method according to claim 30 wherein saidisoprenoid gene is operably linked to an inducible or regulatedpromoter.
 33. A method according to claim 30 wherein said isoprenoidgene is expressed in antisense orientation.
 34. A method according toclaim 30 wherein said isoprenoid gene is disrupted by insertion offoreign DNA into the coding region.
 35. A mutated gene encoding aisoprenoid enzyme having an altered biological activity produced by amethod comprising the steps of: (i) digesting a mixture of nucleotidesequences with restriction endonucleases wherein said mixture comprises:a) a native isoprenoid gene; b) a first population of nucleotidefragments which will hybridize to said native isoprenoid gene; c) asecond population of nucleotide fragments which will not hybridize tosaid native isoprenoid gene; wherein a mixture of restriction fragmentsare produced; (ii) denaturing said mixture of restriction fragments;(iii) incubating the denatured said mixture of restriction fragments ofstep (ii) with a polymerase; (iv) repeating steps (ii) and (iii) whereina mutated isoprenoid gene is produced encoding a protein having analtered biological activity.